Modified polypeptides stabilized in a desired conformation and methods for producing same

ABSTRACT

The present invention provides a method for stabilizing a protein in a desired conformation by introducing at least one disulfide bond into the polypeptide. Computational design is used to identify positions where cysteine residues can be introduced to form a disulfide bond in only one protein conformation, and therefore lock the protein in a given conformation. Accordingly, antibody and small molecule therapeutics are selected that are specific for the desired protein conformation. 
     The invention also provides modified integrin I-domain polypeptides that are stabilized in a desired conformation. The invention further provides screening assays and therapeutic methods utilizing the modified integrin I-domains of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/945,265, filed Aug. 31, 2001, now U.S. Pat. No. 7,160,541, whichclaims priority to U.S. Provisional Patent Application No. 60/229,700.filed on Sep. 1, 2000, all of which are hereby incorporated herein intheir entirety by reference.

GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT

This work was funded in whole or in part by a grant from the NationalInstitutes of Health pursuant to Grant Nos. CA31798 and CA31799. Thefederal government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

The integrin family of adhesion molecules are noncovalently-associatedα/β heterodimers. To date, at least fourteen different integrin αsubunits and eight different β subunits have been reported (Hynes, R O(1992) Cell 69:1–25). Lymphocyte function-associated antigen-1 (LFA-1)is a member of the leukocyte integrin subfamily. Members of theleukocyte integrin subfamily share the common β2 subunit (CD18) but havedistinct α subunits, αL (CD11a), αM (CD11b), αX (CD11c) and αd forLFA-1, Mac-1, p150.95 and αd/β2, respectively (Springer, T A (1990)Nature 346:425–433; Larson, R S and Springer, T A (1990) Immunol Rev114:181–217; Van der Vieren, M et al. (1995) Immunity 3:683–690). Theleukocyte integrins mediate a wide range of adhesive interactions thatare essential for normal immune and inflammatory responses.

Both integrin α and β subunits are type I transmembrane glycoproteins,each with a large extracellular domain, a single transmembrane regionand a short cytoplasmic tail. Several structurally distinct domains havebeen identified or predicted in the α and β subunit extracellulardomains.

The N-terminal region of the integrin α subunits contains seven repeatsof about 60 amino acids each, and has been predicted to fold into a7-bladed β-propeller domain (Springer, T A (1997) Proc Natl Acad Sci USA94:65–72). The leukocyte integrin α subunits, the α1, α2, α10, α11, andαE subunits contain an inserted domain or I-domain of about 200 aminoacids (Larson, R S et al. (1989) J Cell Biol 108:703–712; Takada, Y etal. (1989) EMBO J 8:1361–1368; Briesewitz, R et al. (1993) J Biol Chem268:2989–2996; Shaw, S K et al. (1994) J Biol Chem 269:6016–6025;Camper, L et al. (1998) J Biol Chem 273:20383–20389). The I-domain ispredicted to be inserted between β-sheets 2 and 3 of the β-propellerdomain. The three dimensional structure of the αM, αL, α1 and α2I-domains has been solved and shows that it adopts thedinucleotide-binding fold with a unique divalent cation coordinationsite designated the metal ion-dependent adhesion site (MIDAS) (Lee, J-O,et al. (1995) Structure 3:1333–1340; Lee, J-O, et al. (199S) Cell80:631–638; Qu, A and Leahy, D J (1995) Proc Natl Acad Sci USA92:10277–10281; Qu, A and Leahy, D J (1996) Structure 4:931–942; Emsley,J et al. (1997) J Biol Chem 272:28512–28517; Baldwin, E T et al. (1998)Structure 6:923–935; Kallen, J et al. (1999) J Mol Biol 292:1–9). TheC-terminal region of the αM subunit has been predicted to fold into aβ-sandwich structure (Lu, C et al. (1998) J Biol Chem 273:15138–15147).

The integrin β subunits contain a conserved domain of about 250 aminoacids in the N-terminal portion, and a cysteine-rich region in theC-terminal portion. The β conserved domain, or I-like domain, has beenpredicted to have an “I-domain-like” fold (Puzon-McLaughlin, W andTakada, Y (1996) J Biol Chem 271:20438–20443; Tuckwell, D S andHumphries, M J (1997) FEBS Lett 400: 297–303; Huang, C et al. (2000) JBiol Chem 275:21514–24). The C-terminal Cys-rich region of the βsubunits appears to be important in the regulation of integrin function,as a number of activating antibodies to the β1, β2 and β3 subunits bindto this region (Petruzzelli, L et al. (1995) J Immunol 155:854–866;Robinson, M K et al. (1992) J Immunol 148:1080–1085; Faull, R J et al.(1996) J Biol Chem 271:25099–25106; Shih, D T et al. (1993) J Cell Biol122:1361–1371; Du, X et al. (1993) J Biol Chem 268:23087–23092).

Electron microscopic images of integrins reveal that the N-terminalportions of the α and β subunits fold into a globular head that isconnected to the membrane by two rod-like tails about 16 nm longcorresponding to the C-terminal portions of the α and β extracellulardomains (Nermut, M V et al. (1988), EMBO J 7:4093–4099; Weisel, J W etal. (1992) J Biol Chem 267:16637–16643; Wippler, J et al. (1994) J BiolChem 269: 8754–8761).

LFA-1 is expressed on all leukocytes and is the receptor for three Igsuperfamily members, intercellular adhesion molecule-1, -2 and -3)(Marlin, S D et al. (1987) Cell 51:813–819; Staunton, D E et al. (1989)Nature 339:61–64; de Fougerolles, et al. (1991) J Exp Med 174: 253–267).Substantial data indicate that the I-domain of LFA-1 is critical forinteraction with ligands. Mutagenesis studies have shown that residuesM140, E146, T175, L205, E241, T243, S245 and K263 in the I-domain areimportant for ligand binding (Huang, C et al. (1995) J Biol Chem270:19008–19016; Edwards, C P et al. (1998) J Biol Chem273:28937–28944). These residues are located on the surface of theI-domain surrounding the Mg²⁺ ion, defining a ligand binding interfaceon the upper surface of the I-domain. The importance of the I-domain inligand binding is further underscored by mAb blocking studies. A largenumber mAbs that inhibit LFA-1 interaction with its ligands map to theI-domain (Randi, A M et al. (1994) J Biol Chem 269:12395–12398; Champe,M et al. (1995) J Biol Chem 270:1388–1394; Huang, C et al. (1995) J BiolChem 270:19008–19016; Edwards, C P et al. (1998) J Biol Chem273:28937–28944). Two groups have recently shown that I-domain deletedLFA-1 lacks ligand recognition and binding ability, furtherdemonstrating the role of the I-domain in LFA-1 function (Leitinger, Bet al. (2000) Mol Biol Cell 11, 677–690; Yalamanchili, P et al. (2000) JBiol Chem 275:21877–82). The I-domains of other I-domain containingintegrins have also been implicated in ligand binding (Diamond, M S(1993) J Cell Biol 120:545556; Michishita, M et al. (1993) Cell72:857–867; Muchowski, P J et al. (1994) J Biol Chem 269:26419–26423;Zhou, L et al. (1994) J Biol Chem 269:17075–17079; Ueda, T et al. (1994)Proc Natl Acad Sci USA 91:10680–10684; Kamata, T et al. (1994) J BiolChem 269:96599663; Kern, A et al. (1994) J Biol Chem 269:22811–22816).

Binding of LFA-1 to ICAMs requires LFA-1 activation. LFA-1 can beactivated by signals from the cytoplasm, called “inside-out” signaling(Diamond, M S et al. (1994) Current Biology 4:506–517). Divalent cationsMn²⁰⁺, Mg²⁺ and Ca²⁺ can directly modulate ligand-binding function ofLFA-1 (Dransfield, I et al. (1989) EMBO J 8:3759–3765; Dransfield, I etal. (1992). J Cell Biol 116:219–226; Stewart, M P et al. (1996) JImmunol 156:1810–1817). In addition, LFA-1 can be activated by certainmAbs that bind the extracellular domains of the αL or β2 subunit(Keizer, G D et al. (1988) J Immunol 140:1393–1400; Robinson, M K et al.(1992) J Immunol 148:1080–1085; Andrew, D et al. (1993) Eur J Immunol23:2217–2222; Petruzzelli, L et al. (1995) J Immunol 155:854–866). Themolecular mechanism for integrin activation is not yet well understood.It has been proposed that intramolecular conformational changesaccompanying integrin activation increase integrin affinity for ligand,and this is supported by the existence of antibodies that only recognizeactivated integrins (Dransfield, I et al. (1989) EMBO J 8:3759–3765;Diamond, M S et al. (1993) J Cell Biol 120: 545–556; Shattil, S J et al.(1985) J Biol Chem 260:11107–11114). One of such antibodies CBRLFA-1/5binds to the Mac-1 I-domain very close to the ligand binding site(Oxvig, C et al. (1999) Proc Natl Acad Sci USA 96:2215–2220), providingfurther evidence that the I-domain itself undergoes conformationalchanges with activation.

Two different crystal forms of the Mac-1 I-domain have been obtained,and it has been hypothesized that the two structures represent the“active” and “inactive” conformation, respectively (Lee, J-O et al.(1995) Structure 3, 1333–1340; Lee, J-O et al. (1995) Cell 80:631–638).In the “active” form, crystallized with Mg²⁺, a glutamate from aneighboring I-domain provides the sixth metal coordination site, whilein the “inactive” conformation, complexed with Mn²⁺, a water moleculecompletes the metal coordination sphere. The change in metalcoordination is linked to a large shift of the C-terminal α-helix; inthe putative “active” conformation, the C-terminal helix moves 10 Å downthe body of the I-domain (Lee, J-O et al. (1995) Structure 3:1333–1340).Results from epitope mapping of mAb CBRM-1/5 that only recognizesactivated Mac-1 have suggested that the conformational differences arephysiologically (Oxvig, C et al. (1999) Proc Natl Acad Sci USA96:2215–2220). The crystal and NMR structures of the LFA-1 I-domain havea conformation similar to the putative “inactive” conformation of theMac-1 I-domain (Qu, A et al. (1995) Proc Natl Acad Sci USA92:10277–10281; Qu, A (1996) Structure 4:, 931–942; Kallen, J et al.(1999) J Mol Biol 292:1–9; Legge, G B et al. (2000) J Mol Biol295:1251–1264).

In addition to integrins, many pharmaceutically important proteins existin two alternative three-dimensional structures, referred to asconformations or conformers. Often these proteins have importantsignaling functions, such as small G proteins, trimeric G protein asubunits, tyrosine kinases, and G protein-coupled receptors. Typically,one of these conformations and not the other is enzymatically active orhas effector functions. Therefore, antibody or small moleculetherapeutics that are specific for a protein in a particularconformation, for example, the active conformation, would have greatadvantages over non-selective alternatives.

SUMMARY OF THE INVENTION

Computational design can be used to introduce a disulfide bond into aprotein or polypeptide such that the molecule is stabilized in a desiredconformation. Accordingly, antibodies, e.g., anti-LFA-1 antibodies, orsmall molecule therapeutics that are specific for a desired proteinconformation, e.g., an “open” or active conformation or a “closed” orinactive conformation can be identified.

The invention pertains to methods for stabilizing a polypeptide, e.g., apolypeptide comprising a functional domain of a protein, in a desiredconformation. The method comprises introducing at least one disulfidebond into the polypeptide such that the polypeptide is stabilized in adesired conformation. In a preferred embodiment the disulfide bond isformed by the introduction of at least one cysteine substitution intothe amino acid sequence of the polypeptide. In another embodiment, thedistance between the Cβ carbons in the disulfide bond is in the range of3.00–8.09 Å. In another embodiment, the distance between the Cβ carbonsin the disulfide bond is in the range of 3.41–7.08 Å.

Computational design can be used to introduce a disulfide bond into aprotein or polypeptide such that the molecule is stabilized in a desiredconformation. Accordingly, antibody or small molecule therapeutics thatare specific for a desired protein conformation can be identified.

The method of the invention is widely applicable to a variety ofbiologically and pharmaceutically important proteins that exist in twodifferent three-dimensional conformations, including an integrinsubunit, a small G protein, a heterotrimeric G protein alpha subunit, atyrosine kinase, a G protein-coupled receptor, an enzyme underallosteric control, a zymogen, complement C3, complement C4, andfibrinogen. In a preferred embodiment, the polypeptide is an integrinI-domain polypeptide.

In another aspect, the invention provides a modified integrin I-domainpolypeptide that is stabilized in a desired conformation by theintroduction of at least one disulfide bond. In one embodiment, amodified integrin I-domain is encoded by an amino acid sequencecontaining at least one cysteine substitution as compared to thewild-type sequence, such that a disulfide bond is formed. In anotherembodiment, the distance between the Cβ carbons of the residues that aresubstituted for cysteines is in the range of 3.00–8.09 Å. In yet anotherembodiment, the distance between the Cβ carbons in the disulfide bond isin the range of 3.41–7.08 Å.

In one embodiment, a modified integrin I-domain polypeptide of theinvention is derived from an I-domain of an integrin α subunit, forexample, α1, α2, α10, α11, αD, αE, αL (CD11a), αM (CD11b), and αX(CD11c). For example, in one embodiment of the invention, a modifiedintegrin I-domain polypeptide is derived from the I-domain of the humanαL subunit and contains amino acid substitutions K287C/K294C,E284C/E301C, L161C/F299C, K160C/F299C, L161C/T300C, or L289C/K294C. Inanother embodiment of the invention, a modified integrin I-domainpolypeptide is derived from the I-domain of the human αM subunit andcontains amino acid substitutions Q163C/Q309C, D294C/Q311C, orQ163C/R313C.

In a preferred embodiment, a modified integrin I-domain polypeptide ofthe invention is stabilized in the open conformation. In anotherembodiment, a modified integrin I-domain polypeptide of the invention isstabilized in the closed conformation. In another embodiment, a modifiedintegrin I-domain binds ligand with high affinity. In yet anotherembodiment, a modified integrin I-domain polypeptide is operativelylinked to a heterologous polypeptide.

In a related aspect, the invention provides isolated nucleic acidmolecules which encode a modified integrin I-domain polypeptide of theinvention.

The modified integrin I-domain polypeptides, and/or biologically activeor antigenic fragments thereof, are useful, for example, as reagents ortargets in assays applicable to the treatment and/or diagnosis ofintegrin-mediated disorders.

Accordingly, in one aspect, the invention provides an antibody, or anantigen binding fragment thereof, which selectively binds to a modifiedintegrin I-domain in the open conformation. In another aspect, theinvention provides an antibody, or an antigen binding fragment thereof,which selectively binds to an integrin I-domain polypeptide in the openconformation, an integrin I-domain polypeptide in the closedconformation, or a modified integrin I-domain polypeptide. In oneembodiment, the antibody binds to an activation specific epitope on theintegrin I-domain. In another embodiment, the antibody blocks aninteraction between an integrin and a cognate ligand. In one embodiment,the antibody is an anti-LFA-1 antibody, or an antigen binding fragmentthereof, e.g., an anti-LFA-1 antibody which reacts with or binds an openor closed conformation of an LFA-1 polypeptide, or a modified LFA-1I-domain integrin polypeptide, or fragment thereof.

In another aspect the invention provides a method for identifying amodulator of integrin activity comprising assaying the ability of a testcompound to bind to a modified integrin I-domain polypeptide which isstabilized in the open conformation. In another embodiment, theinvention provides a method for identifying a compound capable ofmodulating the interaction of an integrin and a cognate ligand whereinbinding of a ligand to a modified integrin I-domain polypeptide which isstabilized in the open conformation is assayed in the presence andabsence of a test compound.

In another aspect, the invention provides a composition comprising amodified integrin I-domain polypeptide or an anti-integrin I-domainantibody (or an antigen binding fragment thereof), such composition canfurther include a pharmaceutically acceptable carrier.

In yet another aspect, the invention pertains to methods for treating orpreventing an integrin-mediated disorder (e.g., an inflammatory orautoimmune disorder) in a subject, or for inhibiting the binding of anintegrin to a cognate ligand in a subject comprising administering to asubject a therapeutically effective amount of a modified integrinI-domain polypeptide stabilized in the open conformation or an antibody(or antigen binding fragment thereof) which selectively binds to anintegrin I-domain in the open conformation. In one embodiment, theantibody is an LFA-1 antibody, or an antigen binding fragment thereof,which specifically reacts with or binds an LFA-1 I-domain in the openconformation or specifically reacts with or binds a modified LFA-1I-domain polypeptide. In a preferred embodiment, the modified integrinI-domain polypeptide binds ligand with high affinity. In anotherpreferred embodiment, the modified integrin I-domain polypeptide fortherapeutic use is a soluble polypeptide, e.g., a fusion protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a stereodiagram of the high affinity model of the LFA-1 Idomain, with mutations to introduce a disulfide bond. The model wasprepared using segments of the putative high affinity Mac-1 I domainstructure and a putative low affinity LFA-1 I domain structure astemplates. The K287C and K294C mutations were included in the model. Thesidechains and disulfide bond of C287 and C294 are shown in yellow. TheMg²⁺ ion of the MIDAS is shown as a gold sphere. Sidechains of residuesimportant in binding to ICAM-1 and ICAM-2 are shown with rose-pinksidechains and yellow sulfur, red oxygen, and blue nitrogen atoms. Theseresidues, defined as important in species-specific binding to ICAM-1(Huang, C and Springer, T A (1995) J Biol Chem 270:19008–19016) or by atleast a 2-fold effect on binding to ICAM-1 or ICAM-2 upon mutation toalanine (Edwards, C P et al., (1998) J Biol Chem 273:28937–28944), areM140, E146, T175, L205, E241, T243, S245, and K263. Note that theseresidues surround the Mg²⁺ ion, and are distant from the disulfide.Prepared with RIBBONS (Carson, M (1997) Methods in Enzymology, R M Sweetand C W Carter eds., Academic Press pp. 493–505).

FIG. 2 depicts predicted disulfide bonds that are selective for highaffinity or low affinity conformers of the LFA-1 I domain. TheK287C/K294C mutation (Panels A, C) and L289C/K294C mutation (Panels B,D) were modeled in both high affinity (Panels A, B) and low affinity(Panels C, D) I domain conformers. Residues 254 to 305 of the models areshown. The four models were superimposed using residues not involved inconformational shifts and were used in exactly the same orientation forfigure preparation. Therefore, the downward shift in the α6 helix inpanels A and B compared to panels C and D is readily apparent. Theremodeling of the loop connecting α6 and α6 is accompanied by a reversalin the orientation of the sidechain of residue 289 (panel B compared topanel D). Prepared with RIBBONS.

FIG. 3 depicts the cell surface expression of LFA-1 cysteinesubstitution mutants on 293T transient transfectants (Panel A), and K562stable transfectants (Panel B) as determined by flow cytometric analysisusing monoclonal antibody TS2/4 (shaded histogram) to αL in αL/β2complex, or the nonbinding antibody X63 (open histogram). Numbers in theparentheses are clone numbers of the K562 stable transfectants.

FIG. 4 depicts the binding of LFA-1 transfectants to immobilized ICAM-1.Panel A, 293T transient transfectants, and Panels B and C, K562 stabletransfectants. In Panels A and B, binding of the transfectants toimmobilized ICAM-1 was determined in L15 medium containing Ca²⁺ and Mg²⁺in the presence or absence (control) of the activating antibodyCBRLFA-1/2 at 10 μg/ml. In Panel C, the binding assay was performed inTBS, pH7.5 supplemented with divalent cations or EDTA as indicated.Numbers in the parentheses are clone numbers of the K562 stabletransfectants. Results are mean±SD of triplicate samples andrepresentative of at least three experiments.

FIG. 5 depicts the binding of soluble ICAM-1-IgA fusion protein to K562transfectants that express wild-type LFA-1, the predicted high-affinitymutant K287C/K294C, or mutant L289C/K294C as assessed by flow cytometricanalysis. Mean fluorescent intensity of ICAM-1-IgA binding is indicatedon the upper right corner of the histogram plot. Numbers in theparentheses are clone numbers of the K562 stable transfectants. Resultsare representative of three experiments.

FIG. 6 depicts the inhibitory activity of lovastatin on ligand bindingby cells expressing activated wild-type and high affinity (K287C/K294C)LFA-1.

FIG. 7 depicts the cell surface expression of the isolated LFA-1I-domains. The wild-type αL I-domain and the mutant K287C/K294C andL289C/K294C I-domains were expressed on the surface of the K562transfectants by the PD GFR transmembrane domain. The level of cellsurface I-domain was determined by flow cytometry using monoclonalantibody TS1/22 to the I-domain (shaded histogram). Binding of thecontrol mAb X63 is shown as open histograms. Mean fluorescent intensityof TS1/22 binding was indicated on the upper right corner of thehistogram plot. Results of two individual clones (#1 and #2) from eachI-domain transfectants are shown.

FIG. 8 depicts the ligand binding activity of the cell surface expressedLFA-1 I-domains. Panel A, Binding of K562 transfectants to immobilizedICAM-1 in the presence or absence of DTT. Binding was performed in thepresence (white bar) or absence (black bar) of DTT. Panel B, Effect ofdivalent cations on binding of K562 transfectants to ICAM-1. Binding wasperformed in the presence of Mn²⁺ (black bar), Mg²⁺ (shaded bar) or EDTA(white bar). In Panels A and B, two clones (#1 and #2) of thetransfectants expressing the wild-type I-domain or mutant I-domain weretested. Panel C, Effect of LFA-1 blocking antibodies on binding of theK287C/K294C I-domain to ICAM-1. Results are mean±SD of triplicatesamples and representative of 3 experiments.

FIG. 9 depicts the surface plasmon resonance sensograms by BIAcore™recording the interaction of the open (K287C/K294C) or wild-typeI-domain with ligands, ICAM-1 (Panels A and B), ICAM-2 (Panels C and D),and ICAM-3 (Panels E and F).

FIG. 10 depicts the inhibition of LFA-1-dependent adhesion in vitro bythe open αL I-domain. Panel A depicts the adhesion of K562 stabletransfectants expressing wild-type LFA-1 to immobilized ICAM-1 in thepresence of soluble wild-type (closed circles) or open (K287C/K294C)I-domain (open circles); Panel B depicts the homotypic aggregation ofthe murine EL-4 T lymphoma cell line in the presence of solublewild-type (closed circles) or open (K287C/K294C) I-domain (opencircles).

FIG. 11 depicts the expression and ligand binding activity of the Mac-1cysteine substitution mutants in transiently transfected 293T cells.Panel A, binding of monoclonal antibodies CBRM1/32 (open bars) andCBRM1/5 (black bars) to intact Mac-1 I-domain mutants. Panel B, adhesionof 293T transient transfectants expressing intact Mac-1 cysteinesubstitution mutants to iC3b coated on plastic. Panel C, adhesion of293T transient transfectants expressing isolated Mac-1mutant I-domainsto iC3b ligand in the presence (black bars) or absence (open bars) ofantibody CBRM1/5.

FIG. 12 depicts the expression and ligand binding activity of the Mac-1cysteine substitution mutants in K562 stable transfectants. Panel A,representative histogram showing binding of monoclonal antibodiesCBRM1/32 and CBRM1/5 to intact Mac-1 I-domain mutants as assessed byflow cytometry. Mean fluorescent intensity is indicated in the upperright hand corner of the histogram plot. Panel B, adhesion of K562stable transfectants expressing intact Mac-1 cysteine substitutionmutants to iC3b coated on plastic. Panel C, adhesion of K562 stabletransfectants expressing isolated Mac-1 I-mutant I-domains to iC3bligand. Adhesion was assayed in the presence (black bars) or absence(open bars) of monoclonal antibody CBRM1/5, or in the presence of 10 mMDTT (gray bars).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on a method forstabilizing a polypeptide in a desired conformation by introducing atleast one disulfide bond into the polypeptide. In one embodiment, basedon NMR or crystal structures of specific protein conformations,computational design is used to introduce a disulfide bond that locksthe protein in a particular conformation. As used herein, a“conformation” or “conformer” refers to the three dimensional structureof a protein. A “desired” conformation includes a protein conformationthat is conducive to a particular use of the polypeptide, e.g., aconformation that supports a particular biological function and/oractivity, or a therapeutic effect. As used herein, the terms“polypeptide” and “protein” are used interchangeably throughout.

In one embodiment, a desired conformation is a protein conformationwhich promotes or activates a biological function and/or activity, e.g.,an open or active conformation. In another embodiment, a desiredconformation is a protein conformation which inhibits or suppresses abiological function and/or activity, e.g., a closed or inactiveconformation.

In particular, the method of the invention includes modeling a protein,or a functional domain thereof, on a template of the desiredthree-dimensional structure of the protein and introducing cysteineswhich are able to form a disulfide bond only in the desired conformationof the protein, thus stabilizing the protein in that particularconformation. The protein can be any protein, or domain thereof, forwhich a three dimensional structure is known or can be generated, and ispreferably a protein that exists in two different conformations.Computational algorithms for designing and/or modeling proteinconformations are described, for example, in WO 98/47089. The SSBONDprogram (Hazes, B and Dijkstra, B W (1988) Protein Engineering2:119–125) can be used to identify positions where disulfide bonds canbe introduced in a protein structure by mutating appropriatelypositioned pairs of residues to cysteine.

Disulfide bond formation occurs between two cysteine residues that areappropriately positioned within the three-dimensional structure of apolypeptide. In one embodiment of the invention, a polypeptide isstabilized in a desired conformation by introducing at least onecysteine substitution into the amino acid sequence such that a disulfidebond is formed. The introduction of a single cysteine substitution isperformed in circumstances in which an additional cysteine residue ispresent in the native amino acid sequence of the polypeptide at anappropriate position such that a disulfide bond is formed. In apreferred embodiment, two cysteine substitutions are introduced into theamino acid sequence of the polypeptide at positions that allow adisulfide bond to form, thereby stabilizing the polypeptide in a desiredconformation. In another embodiment, the distance between the Cβ carbonsof the residues that are substituted for cysteine is 3.00–8.09 Å. In yetanother embodiment, the distance between the Cβ carbons in the disulfidebond is in the range of 3.41–7.08 Å.

In one embodiment of the invention, cysteine substitutions areintroduced such that the formation of a disulfide bond is favored onlyin one protein conformation, such that the protein is stabilized in thatparticular conformation.

Preparation of a modified polypeptide of the invention by introducingcysteine substitutions is preferably achieved by mutagenesis of DNAencoding the polypeptide of interest (e.g., an integrin polypeptide).For example, an isolated nucleic acid molecule encoding a modifiedintegrin I-domain polypeptide can be created by introducing one or morenucleotide substitutions into the nucleotide sequence of an integringene such that one or more amino acid substitutions, e.g., cysteinesubstitutions, are introduced into the encoded protein. Mutations can beintroduced into a nucleic acid sequence by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis.

Suitable proteins include, but are not limited to, industrially andtherapeutically important proteins such as: 1) signaling molecules, suchas small G proteins, trimeric G protein alpha subunits, tyrosinekinases, and G protein-coupled receptors; 2) enzymes under allostericcontrol, 3) zymogens that undergo conformational change after activationby proteolytic cleavage, such as the proteases (convertases and factors)of the complement and clotting cascades, and 4) proteolyticallyactivated effector molecules such as complement components C3 and C4,and fibrinogen. In one embodiment, the method of the invention can beused to stabilize a protein in a biologically active conformation, e.g.,a conformation that is enzymatically active or has ligand bindingcapacity and/or effector functions, e.g., an “open” conformation. Inanother embodiment, the method of the invention can be used to stabilizea protein in a biologically inactive conformation, e.g., a conformationthat is enzymatically inactive or does not have ligand binding capacityand/or effector functions, e.g., a “closed” conformation.

Proteins that are stabilized in a particular conformation may find usein, for example, in proteomic screening technologies. In proteomicscreens of tissues and disease states, antibodies, polypeptide, and/orsmall molecules that are specific for, e.g., an active protein conformeror an inactive protein conformer, can be used to assess the activity ofdifferent cellular signaling, metabolic, and adhesive pathways. Thus,associations can be made between specific diseases and the activation ofspecific biochemical and signaling pathways. Furthermore, the inventionrelates to the polypeptides, antibodies, and small molecules identifiedusing the methods described herein and uses for same, e.g., to treat,for example, inflammatory disorders. Conformer-specific reagents canalso be placed on chips and used to screen tissue extracts, or used tostain tissue sections. Furthermore, drugs or antibodies, e.g.,anti-integrin antibodies which specifically recognize a modifiedintegrin I-domain polypeptide, e.g., an anti-LFA-1 antibody whichspecifically recognizes a modified LFA-1 I-domain polypeptide, that areselective for a particular conformer, e.g., an open conformer or aclosed conformer, may provide differential therapeutic effects.Therefore, selective screening assays using a protein stabilized in aparticular conformer can be used to rationally obtain compounds with adesired activity.

Integrins

Integrins exist on cell surfaces in an inactive conformation that doesnot bind ligand. Upon cell activation, integrins change shape(conformation) and can bind ligand. Over 20 different integrinheterodimers (different α and β subunit combinations) exist that areexpressed in a selective fashion on all cells in the body. Afteractivation, integrins bind in a specific manner to protein ligands onthe surface of other cells, in the extracellular matrix, or that areassembled in the clotting or complement cascades. Integrins onleukocytes are of central importance in leukocyte emigration and ininflammatory and immune responses. Ligands for the leukocyte integrinMac-1 (αMβ2) include the inflammation-associated cell surface moleculeICAM-1, the complement component iC3b, and the clotting componentfibrinogen. Ligands for the leukocyte integrin LFA-1 (αLβ2) includeICAM-1, ICAM-2, and ICAM-3. Antibodies to leukocyte integrins can blockmany types of inflammatory and auto-immune diseases, by, e.g.,modulating, e.g., inhibiting, for example, cell to cell interactions orcell to extracellular matrix interactions. Integrins on platelets areimportant in clotting and in heart disease; approved drugs include theantibody abciximab (Reopro™) and the peptide-like antagonisteptifibatide (Integrilin™). Integrins on connective tissue cells,epithelium, and endothelium are important in disease states affectingthese cells. They regulate cell growth, differentiation, wound healing,fibrosis, apoptosis, and angiogenesis. Integrins on cancerous cellsregulate invasion and metastasis.

To antagonize integrins, drugs are needed that bind to the active,ligand-binding conformation. Most antibodies bind to both the active andinactive conformations, since only a small portion of the surface of theintegrin molecule changes shape. It is desirable that antibodies bindonly to the active integrin conformation, e.g., the “open” conformation,because binding to the inactive conformation can lead to side reactions,generation of anti-idiotypic antibodies, and result in clearance of theantibody and, thus, requires much higher doses to be administered.

The methods described herein have been successfully used to introducedisulfide bonds into the I domains of the integrins, e.g., LFA-1 andMac-1. Accordingly, in another aspect, the invention provides a modifiedintegrin I-domain polypeptide containing at least one disulfide bond,such that said modified I-domain polypeptide is stabilized in a desiredconformation. A modified integrin I-domain polypeptide of the inventionmay be derived from an I-domain of an integrin α subunit including α1,α2, α10, α11, αD, αE, αL (CD11a), αM (CD11b) and αX (CD11c).

As used herein, a “modified integrin I-domain polypeptide” or “modifiedintegrin polypeptide” includes an integrin I-domain polypeptide that hasbeen altered with respect to the wild-type sequence or the native statesuch that at least one disulfide bond has been introduced into thepolypeptide thereby stabilizing the I-domain in a desired conformation.

The terms “derived from” or “derivative”, as used interchangeablyherein, are intended to mean that a sequence is identical to or modifiedfrom another sequence, e.g., a naturally occurring sequence. Derivativeswithin the scope of the invention include polynucleotide and polypeptidederivatives. Polypeptide or protein derivatives include polypeptide orprotein sequences that differ from the sequences described or known inamino acid sequence, or in ways that do not involve sequence, or both,and still preserve the activity of the polypeptide or protein.Derivatives in amino acid sequence are produced when one or more aminoacid is substituted with a different natural amino acid, an amino acidderivative or non-native amino acid. In certain embodiments proteinderivatives include naturally occurring polypeptides or proteins, orbiologically active fragments thereof, whose sequences differ from thewild-type sequence by one or more conservative amino acid substitutions,which typically have minimal influence on the secondary structure andhydrophobic nature of the protein or peptide. Derivatives may also havesequences which differ by one or more non-conservative amino acidsubstitutions, deletions or insertions which do not abolish thebiological activity of the polypeptide or protein.

Conservative substitutions (substituents) typically include thesubstitution of one amino acid for another with similar characteristics(e.g., charge, size, shape, and other biological properties) such assubstitutions within the following groups: valine, glycine; glycine,alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. The non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan andmethionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

In other embodiments, derivatives with amino acid substitutions whichare less conservative may also result in desired derivatives, e.g., bycausing changes in charge, conformation and other biological properties.Such substitutions would include, for example, substitution ofhydrophilic residue for a hydrophobic residue, substitution of acysteine or proline for another residue, substitution of a residuehaving a small side chain for a residue having a bulky side chain orsubstitution of a residue having a net positive charge for a residuehaving a net negative charge. When the result of a given substitutioncannot be predicted with certainty, the derivatives may be readilyassayed according to the methods disclosed herein to determine thepresence or absence of the desired characteristics. The polypeptides andproteins of this invention may also be modified by various changes suchas insertions, deletions and substitutions, either conservative ornonconservative where such changes might provide for certain advantagesin their use.

In a preferred embodiment, a modified integrin I-domain polypeptide isstabilized in the open conformation, and binds ligand with highaffinity.

In one embodiment, a modified integrin I-domain polypeptide of theinvention is encoded by an amino acid sequence containing at least onecysteine substitution, and preferably two cysteine substitutions, ascompared to the wild-type sequence. In another embodiment, the distancebetween the Cβ carbons of the residues that are substituted forcysteines is in the range of 3.00–8.09 Å, e.g., as predicted by proteinmodeling. In a further embodiment, the distance between the Cβ carbonsin the disulfide bond is in the range of 3.41–7.08 Å.

The introduction of cysteine residues at appropriate positions withinthe amino acid sequence of the I-domain polypeptide allows for theformation of a disulfide bond that stabilizes the domain in a particularconformation, e.g., an active “open” conformation, or an inactive“closed” conformation. For example, the αL K287C/K294C, E284C/E301C,L161C/F299C, K160C/F299C, L161C/T300C, and L289C/K294C mutants, and theαM Q163C/Q309C and D294C/Q311C mutants are stabilized in the “open”conformation that bind ligand with high or intermediate affinity,whereas the αL L289C/K294C mutant and the αM Q163C/R313C mutants arestabilized in an inactive or “closed” conformation that does not bindligand. The affinity of E284C/E301C is nearly comparable to that ofK287C/K294C, e.g., high-affinity. The affinity of L161C/F299C,K160C/F299C, and L161C/T300C are significantly higher than wild-type,but 20–30 times lower than high-affinity αL I-domain, K287C/K294C.L161C/F299C, K160C/F299C, and L161C/T300C are referred to herein asintermediate affinity αL I-domains.

In one embodiment, the invention provides a modified integrin I-domainwhich is comprised within an integrin α subunit, and which may befurther associated with an integrin β subunit. In another embodiment, amodified integrin I-domain polypeptide of the invention is a solublepolypeptide. Furthermore, the invention provides a modified integrinI-domain polypeptide which is operatively linked to a heterologouspolypeptide.

A model of the I-like domain of the integrin β-subunit that is supportedby experimental data (Huang, C et al. (2000) J Biol Chem 275:21514–24)has also been made. The data confirm the location of the key C-terminalα-helix that undergoes the dramatic 10 Å conformational movement in Idomains. The I and I-like domains align well in this region.Accordingly, in another aspect, the invention provides a modifiedintegrin I-like domain polypeptide containing at least one disulfidebond, such that said modified I-like domain polypeptide is stabilized ina desired conformation.

In a preferred embodiment, a modified integrin I-like domain polypeptideis stabilized in the open conformation, and binds ligand with highaffinity. In one embodiment, a modified integrin I-like domainpolypeptide of the invention is encoded by an amino acid sequencecontaining at least one cysteine substitution, and preferably twocysteine substitutions, as compared to the wild-type sequence.

In one embodiment, the invention provides a modified integrin I-likedomain which is comprised within an integrin β subunit, and which may befurther associated with an integrin α subunit. In another embodiment, amodified integrin I-like domain polypeptide of the invention is asoluble polypeptide. Furthermore, the invention provides a modifiedintegrin I-like domain polypeptide which is operatively linked to aheterologous polypeptide.

Integrins are key targets in many diseases. Accordingly, isolated highaffinity I-domains of the invention, as well as antibodies, or smallmolecule antagonists selective for activated leukocyte integrins can beused to modulate, e.g., inhibit or prevent, autoimmune and inflammatorydisease, transplant rejection, and ischemia/reperfusion injury as inhypovolemic shock, myocardial infarct, and cerebral shock. Furthermore,co-crystals of high affinity I domains bound to natural ligands and/orsmall molecule antagonists can readily be made, which will enablecomputational drug design, and advance modification and improvement ofdrug development candidates.

Accordingly, the invention provides a method for identifying a modulatorof integrin activity comprising assaying the ability of a test compoundto bind to a modified integrin I-domain polypeptide which is stabilizedin the open conformation. In another embodiment, the invention providesa method for identifying a compound capable of modulating theinteraction of an integrin and a cognate ligand wherein binding of aligand to a modified integrin I-domain polypeptide which is stabilizedin the open conformation is assayed in the presence and absence of atest compound.

The invention also provides a composition comprising a modified integrinI-domain polypeptide or an anti-integrin antibody, e.g., an anti-LFA-1antibody (or an antigen binding fragment thereof) which selectivelybinds to a modified integrin I-domain, e.g., an I-domain in the openconformation, and a pharmaceutically acceptable carrier. Thecompositions of the invention are used in therapeutic methods of theinvention. For example, the invention provides methods for treating orpreventing an integrin-mediated disorder (e.g., an inflammatory orautoimmune disorder) in a subject, or for inhibiting the binding of anintegrin to a cognate ligand in a subject comprising administering to atherapeutically effective amount of a modified integrin I-domainpolypeptide stabilized in the open conformation or anti-integrinantibody (or an antigen binding fragment thereof) which selectivelybinds to an integrin I-domain in the open conformation. In a preferredembodiment, the modified integrin I-domain polypeptide binds ligand withhigh affinity. In another preferred embodiment, the modified integrinI-domain polypeptide for therapeutic use is a soluble polypeptide, e.g.,a fusion protein.

As used herein, an integrin mediated disorder includes, for example, aninflammatory or immune system disorder, and/or a cellular proliferativedisorder. Examples of integrin-mediated disorders include myocardialinfarction, stroke, restenosis, transplant rejection, graft versus hostdisease or host versus graft disease, and reperfusion injury. Aninflammatory or immune system disorder includes, but is not limited toadult respiratory distress syndrome (ARDS), multiple organ injurysyndromes secondary to septicemia or trauma, viral infection,inflammatory bowel disease, ulcerative colitis, Crohn's disease,leukocyte adhesion deficiency II syndrome, thermal injury, hemodialysis,leukapheresis, peritonitis, chronic obstructive pulmonary disease, lunginflammation, asthma, acute appendicitis, dermatoses with acuteinflammatory components, wound healing, septic shock, acuteglomerulonephritis, nephritis, amyloidosis, reactive arthritis,rheumatoid arthritis, chronic bronchitis, Sjorgen's syndrome,sarcoidosis, scleroderma, lupus, polymyositis, Reiter's syndrome,psoriasis, dermatitis, pelvic inflammatory disease, inflammatory breastdisease, orbital inflammatory disease, immune deficiency disorders(e.g., HIV, common variable immunodeficiency, congenital X-linkedinfantile hypogammaglobulinemia, transient hypogammaglobulinemia,selective IgA deficiency, necrotizing enterocolitis, granulocytetransfusion associated syndromes, cytokine-induced toxicity, chronicmucocutaneous candidiasis, severe combined immunodeficiency), autoimmunedisorders, and acute purulent meningitis or other central nervous systeminflammatory disorders.

A “cellular proliferative disorder” includes those disorders that affectcell proliferation, activation, adhesion, growth, differentiation, ormigration processes. As used herein, a “cellular proliferation,activation, adhesion, growth, differentiation, or migration process” isa process by which a cell increases in number, size, activation state,or content, by which a cell develops a specialized set ofcharacteristics which differ from that of other cells, or by which acell moves closer to or further from a particular location or stimulus.Disorders characterized by aberrantly regulated growth, activation,adhesion, differentiation, or migration. Such disorders include cancer,e.g., carcinoma, sarcoma, lymphoma or leukemia, examples of whichinclude, but are not limited to, breast, endometrial, ovarian, uterine,hepatic, gastrointestinal, prostate, colorectal, and lung cancer,melanoma, neurofibromatosis, adenomatous polyposis of the colon, Wilms'tumor, nephroblastoma, teratoma, rhabdomyosarcoma; tumor invasion,angiogenesis and metastasis; skeletal dysplasia; hematopoietic and/ormyeloproliferative disorders.

Various aspects of the invention are described in further detail in thefollowing subsections.

Modified Integrin I-Domain Polypeptides and Anti-Integrin I-DomainAntibodies

The methods of the invention include the use of isolated, modifiedintegrin polypeptides, and biologically active portions thereof. As usedherein, a modified integrin polypeptide includes a modified I-domainpolypeptide and a modified I-like domain polypeptide. Modified integrinpolypeptides of the invention include modified integrin I-domain andI-like domain polypeptides that are comprised within an integrin α or βsubunit polypeptide, respectively; soluble modified integrin I-domainand I-like domain polypeptides; and modified integrin I-domain andI-like domain polypeptides that are operatively linked to a heterologouspolypeptide, e.g., fusion proteins.

The cDNAs for multiple human integrin α and β subunit polypeptides havebeen cloned and sequenced, and the polypeptide sequences have beendetermined (see, for example, GenBank Accession Numbers: NM_(—)002203(α2), AF112345 (α10), NM_(—)012211 (α11), NM_(—)005353 (αD),NM_(—)002208 (αE), NM_(—)000887 (αX), NM_(—)000632 (αM), NM_(—)002209(αL), X68742 and P56199 (α1), NM_(—)000211 (β2), NM_(—)000212 (β3),NM_(—)002214 (β8)). In particular, the polypeptide sequences encodinghuman αL and αM are set forth as SEQ ID NO:2 (GenBank Accession No.P20701) and SEQ ID NO:4 (GenBank Accession No. P11215), respectively. Inaddition, the sequences encoding integrin α and β subunit polypeptidesfrom other species are available in the art. Furthermore, as describedpreviously, three dimensional structure of the αM, αL, α1 and α2I-domains has been solved (Lee, J-O, et al. (1995) Structure3:1333–1340; Lee, J-O, et al. (199S) Cell 80:631–638; Qu, A and Leahy, DJ (1995) Proc Natl Acad Sci USA 92:10277–10281; Qu, A and Leahy, D J(1996) Structure 4:931–942; Emsley, J et al. (1997) J Biol Chem272:28512–28517; Baldwin, E T et al. (1998) Structure 6:923–935; Kallen,J et al. (1999) J Mol Biol 292:1–9).

Isolated modified integrin polypeptides of the present inventionpreferably have an amino acid sequence that is sufficiently identical tothe amino acid sequence of a native integrin polypeptide, yet whichcomprise at least one, and preferably two cysteine substitutions, suchthat a disulfide bond is formed that stabilizes the polypeptide in adesired conformation. As used herein, the term “sufficiently identical”refers to an amino acid (or nucleotide) sequence which contains asufficient or minimum number of identical or equivalent (e.g., an aminoacid residue that has a similar side chain) amino acid residues (ornucleotides) to a integrin amino acid (or nucleotide) sequence such thatthe polypeptide shares common structural domains or motifs, and/or acommon functional activity with a native integrin polypeptide. Forexample, amino acid or nucleotide sequences which share at least 30%,40%, or 50%, preferably 60%, more preferably 70%, 75%, 80%, 85% or 90%,91%, 92%, 93%, 94%, 95% or greater identity and share a commonfunctional activity (e.g., an activity of a modified integrin I-domainor I-like domain as described herein) are defined herein as sufficientlyidentical. An integrin I-domain polypeptide may differ in amino acidsequence from the integrin polypeptides disclosed herein due to naturalallelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444–453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two-amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (Comput. Appl. Biosci., 4:11–17 (1988)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4.

As used herein, a “biologically active portion” of a modified integrinpolypeptide (e.g., a modified integrin I-domain polypeptide) includes afragment of a modified integrin polypeptide which retains a modifiedintegrin polypeptide activity. Typically, a biologically active portionof a modified integrin polypeptide comprises at least one domain ormotif with at least one activity of the modified integrin polypeptide,e.g., ligand binding. In a preferred embodiment, biologically activeportions of a modified integrin polypeptide include modified integrinI-domain polypeptides. Biologically active portions of a modifiedintegrin polypeptide may comprise amino acid sequences sufficientlyidentical to or derived from the amino acid sequence of a modifiedintegrin polypeptide, which include less amino acids than the fulllength modified integrin polypeptide, and exhibit at least one activityof a modified integrin polypeptide. Biologically active portions of amodified integrin polypeptide, e.g., a modified I-domain or I-likedomain, can be used as targets for developing agents which modulate aintegrin polypeptide activity, e.g., ligand binding, adhesion, e.g.,cell to cell adhesion or cell to extracellular matrix adhesion, and/orsignaling activity. A biologically active portion of a modified integrinpolypeptide comprises a polypeptide which can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofa modified integrin polypeptide.

In a preferred embodiment, modified integrin polypeptides are producedby recombinant DNA techniques. For example, a modified integrinpolypeptide can be isolated from a host cell transfected with apolynucleotide sequence encoding a modified integrin polypeptide (e.g.,a I-domain polypeptide or a soluble I-domain fusion protein) using anappropriate purification scheme using standard protein purificationtechniques. Alternative to recombinant expression, a modified integrinpolypeptide can be synthesized chemically using standard peptidesynthesis techniques.

An “isolated” or “purified” polypeptide or protein, or biologicallyactive portion thereof is substantially free of cellular material orother contaminating proteins from the source, e.g., the cellular source,from which the modified integrin I-domain polypeptide is derived, orsubstantially free from chemical precursors or other chemicals whenchemically synthesized. The language. “substantially free of cellularmaterial” includes preparations of modified integrin polypeptide inwhich the protein is separated from cellular components of the cellsfrom which it is isolated or recombinantly produced. In one embodiment,the language “substantially free of cellular material” includespreparations of modified integrin polypeptide having less than about 30%(by dry weight) of non-modified integrin polypeptide (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-modified integrin polypeptide, still more preferably lessthan about 10% of non-modified integrin polypeptide, and most preferablyless than about 5% non-modified integrin polypeptide. When the modifiedintegrin polypeptide or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of modified integrin polypeptide inwhich the protein is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein. In oneembodiment, the language “substantially free of chemical precursors orother chemicals” includes preparations of modified integrin polypeptidehaving less than about 30% (by dry weight) of chemical precursors ornon-modified integrin polypeptide chemicals, more preferably less thanabout 20% chemical precursors or non-modified integrin polypeptidechemicals, still more preferably less than about 10% chemical precursorsor non-modified integrin polypeptide chemicals, and most preferably lessthan about 5% chemical precursors or non-modified integrin polypeptidechemicals.

The methods of the invention may also use modified integrin polypeptidesthat are chimeric or fusion proteins. As used herein, a modifiedintegrin “chimeric protein” or “fusion protein” comprises a modifiedintegrin polypeptide operatively linked to a non-modified integrinpolypeptide, e.g., a heterologous polypeptide. In a preferredembodiment, a modified integrin fusion protein comprises at least anI-domain or an I-like domain. Within the fusion protein, the term“operatively linked” is intended to indicate that the modified integrinpolypeptide and the heterologous polypeptide sequences are fusedin-frame to each other. The heterologous polypeptide can be fused to theN-terminus or C-terminus of the modified integrin polypeptide.

For example, in a preferred embodiment, the fusion protein is a modifiedintegrin-I-domain fusion protein in which the Fc region, e.g., thehinge, C1 and C2 sequences, of an immunoglobulin, (e.g., human IgG1) isfused to the C-terminus of the modified integrin sequences. Integrinimmunoglobulin chimeras can be constructed essentially as described inWO 91/08298. Such fusion proteins can facilitate the purification ofrecombinant modified integrin polypeptides. In another embodiment, thefusion protein is a modified integrin I-domain polypeptide fused to aheterologous transmembrane domain, such that the fusion protein isexpressed on the cell surface.

The modified integrin polypeptides and fusion proteins of the inventioncan be incorporated into pharmaceutical compositions and administered toa subject in vivo. In an exemplary embodiment, a soluble modifiedintegrin I-domain polypeptide stabilized in an open, ligand bindingconformation, or fusion protein thereof may be used to modulate integrinactivity (e.g., integrin binding to a cognate ligand) in a subject. Inanother embodiment, a soluble modified integrin I-domain polypeptide orfusion protein may be used to treat an inflammatory or immune systemdisorder, e.g., an autoimmune disorder. In another embodiment, a solublemodified integrin polypeptide or fusion protein may be used to treat acellular proliferative disease. Use of soluble modified integrinpolypeptides and fusion proteins can also be used to affect thebioavailability of a integrin ligand, e.g., ICAM.

Moreover, the modified integrin polypeptides and fusion proteins of theinvention can be used as immunogens to produce anti-integrin antibodiesin a subject, e.g., anti-LFA-1 antibodies, and in screening assays toidentify molecules which modulate integrin activity, and/or modulate theinteraction of a integrin polypeptide with a integrin ligand orreceptor.

Preferably, a modified integrin fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A modifiedintegrin polypeptide-encoding nucleic acid can be cloned into such anexpression vector such that the fusion moiety is linked in-frame to themodified integrin polypeptide.

The methods of the present invention may also include the use ofmodified integrin polypeptides which function as either integrinagonists (mimetics) or as integrin antagonists. An agonist of anintegrin polypeptide can retain substantially the same, or a subset, ofthe biological activities of the naturally occurring form of a integrinpolypeptide. An antagonist of an integrin polypeptide can inhibit one ormore of the activities of a native form of the integrin polypeptide by,for example, competitively modulating an integrin activity. Thus,specific biological effects can be elicited by treatment with a modifiedintegrin polypeptide stabilized in a desired conformation.

An isolated, modified integrin polypeptide, e.g., a modified LFA-1polypeptide, or a portion or fragment thereof, can be used as animmunogen to generate antibodies that bind to a specific conformation ofan integrin, e.g., an integrin I-domain, using standard techniques forpolyclonal and monoclonal antibody preparation (see, generally R. H.Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387–402; M. L. Gefter et al. (1977)Somatic Cell Genet. 3:231–36). Moreover, the ordinarily skilled artisanwill appreciate that there are many variations of such methods whichalso would be useful. Preparation of anti-LFA-1 antibodies is describedin, for example, U.S. Pat. No. 5,622,700, the entire content of which isincorporated herein by this reference.

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which specifically binds(immunoreacts with) an antigen, e.g., an integrin I-domain in an open orclosed conformation, or a modified integrin I-domain, such as an LFA-1I-domain, e.g., an open or closed LFA-1 I-domain or a modified integrinI-domain of LFA-1. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin. Theinvention provides polyclonal and monoclonal antibodies that bind amodified integrin polypeptide e.g., a modified LFA-1 polypeptide, or aportion or fragment thereof. The term “monoclonal antibody” or“monoclonal antibody composition”, as used herein, refers to apopulation of antibody molecules that contain only one species of anantigen binding site capable of immunoreacting with a particular epitopeof a modified integrin polypeptide, e.g., a modified LFA-1 polypeptide,or a portion or fragment thereof. A monoclonal antibody composition thustypically displays a single binding affinity for a particular modifiedintegrin polypeptide, or a portion or fragment thereof with which itimmunoreacts.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-integrin antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with a modified integrin polypeptide,e.g., a modified integrin I-domain stabilized in the open conformation,to thereby isolate immunoglobulin library members that bind to anconformation specific epitope on an integrin polypeptide, e.g., an openconformation. Kits for generating and screening phage display librariesare commercially available (e.g., the Pharmacia Recombinant PhageAntibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™Phage Display Kit, Catalog No. 240612). With regard to screening forphage libraries with integrin I-domains locked in the high affinityconformation with a disulfide bond, note that it should be possible toelute specific phage by adding a reducing agent that breaks thedisulfide and abolishes the high affinity conformation of the I-domain.

Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370–1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81–85; Huse et al. (1989) Science 246:1275–1281;Griffiths et al. (1993) EMBO J 12:725–734; Hawkins et al. (1992) J. Mol.Biol. 226:889–896; Clarkson et al. (1991) Nature 352:624–628; Gram etal. (1992) Proc. Natl. Acad. Sci. USA 89:3576–3580; Garrad et al. (1991)Bio/Technology 9:1373–1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133–4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978–7982; and McCafferty et al. Nature (1990) 348:552–554.

Additionally, recombinant anti-integrin antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,can also be used in the methods of the present invention. Such chimericand humanized monoclonal antibodies can be produced by recombinant DNAtechniques known in the art, for example using methods described inRobinson et al. International Application No. PCT/US86/02269; Akira, etal. European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison et al. European Patent Application173,494; Neuberger et al. PCT International Publication No. WO 86/01533;Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European PatentApplication 125,023; Better et al. (1988) Science 240:1041–1043; Liu etal. (1987) Proc. Natl. Acad. Sci. USA 84:3439–3443; Liu et al. (1987) J.Immunol. 139:3521–3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA84:214–218; Nishimura et al. (1987) Canc. Res. 47:999–1005; Wood et al.(1985) Nature 314:446–449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553–1559); Morrison, S. L. (1985) Science 229:1202–1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552–525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053–4060.

In a preferred embodiment, an anti-integrin antibody of the inventionbinds selectively to an integrin I-domain in the open, high-affinityconformation, e.g., at an epitope that is unique to an activatedintegrin (also referred to herein as an activation specific epitope). Ina preferred embodiment, an anti-integrin antibody of the inventionmodulates (e.g., inhibits) the binding interaction between an activatedintegrin and its cognate ligand. In another embodiment, an anti-integrinantibody inhibits leukocyte adhesion and/or aggregation. In anotherembodiment, an anti-integrin antibody of the invention binds selectivelyto an integrin I-domain in an open conformation, e.g., an LFA-1 I-domainin an open conformation, or a modified integrin I-domain, e.g., amodified I-domain of an LFA-1 molecule.

An anti-integrin antibody (e.g., a monoclonal antibody) can be used inthe methods of the invention to modulate the expression and/or activityof an integrin or an integrin I-domain polypeptide. An anti-integrinantibody can also be used to isolate modified integrin or integrinI-domain polypeptides, e.g., a modified LFA-1 polypeptide, or fusionproteins by standard techniques, such as affinity chromatography orimmunoprecipitation. In another embodiment, an anti-integrin antibodycan be used to remove and/or kill cells expressing activated integrin.Moreover, an anti-integrin antibody can be used to detect integrinpolypeptides in a particular conformation (e.g., an activated integrin),for example, for the localization of stimulated and/or activatedleukocytes. Furthermore, an anti-integrin antibody, e.g., an antibodywhich reacts with or binds an integrin I-domain in an open conformationor a modified integrin I-domain, can be used therapeutically asdescribed herein. Accordingly anti-integrin antibodies can be useddiagnostically to monitor protein levels in blood as part of a clinicaltesting procedure, e.g., to, for example, detect inflammation. Detectioncan be facilitated by coupling (i.e., physically linking) the antibodyto a detectable substance. Examples of detectable substances includevarious enzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Isolated Nucleic Acid Molecules

The invention includes the use of isolated nucleic acid molecules thatencode integrin polypeptides (e.g., a modified integrin I-domainpolypeptide, e.g., a modified integrin I-domain or I-like domainpolypeptide) or biologically active portions thereof.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., eDNA or genomic DNA) and RNA molecules (e.g., mRNA)and analogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. The nucleotide sequences encoding thewild-type human αL and αM polypeptides are set forth as SEQ ID NO:1(GenBank Accession No. NM_(—)002209) and SEQ ID NO:3 (Genbank AccessionNo. J03925), respectively. The isolated nucleic acid molecules of thepresent invention include the nucleotide sequences of SEQ ID NO:1 andSEQ ID NO:3, which encode the modified amino acid sequences of the αLand αM mutants described herein, e.g., identified below in fable 9.Table 9 illustrates the specific nucleotide residues which are alteredto result in the modified αL and αM mutants as described herein. Forexample, the K287C/K294C mutant is a modified αL polypeptide, whereinthere is a change in the amino acid sequence of (SEQ ID NO:2) such thatamino acid residues 287 and 294 are substituted wit cysteine residues.The corresponding wild-type nucleotide sequence, SEQ ID NO:1, ismodified at nucleotide residues 1022–1024 and 1043–1045, respectively.Therefore, as shown in table 9, for the αL K287C/K294C mutant at aminoacid K287, the corresponding nucleotide residues in the wild-type αLnucleic acid sequence (SEQ ID NO:1), nucleotide residues 1022–1024, aremodified from “aaa” to “tgt.” Note that SEQ ID NO:2 and SEQ ID NO:4 arethe amino acid sequences of the precursor proteins, while the numbersystem used herein is based on the mature protein. The precursor proteinof SEQ ID NO:2 includes 25 additional amino acids, as compared with themature protein, while the precursor protein for SEQ ID NO:4 includes 16additional amino acids, as compared to the mature protein. Theadditional amino acids for each protein reside at the beginning of therespective sequences.

TABLE 9 Nucleotide Mutants # sequence αM or αL mutations Amino Acid#Nucleotide WT mutant αL K287C/K294C K287 1022–1024 aaa tgt K2941043–1045 aag tgt E284C/E301C E284 1013–1015 gag tgt E301 1064–1066 gagtgt L161C/F299C L161 644–646 ctc tgt F299 1058–1060 ttc tgt K160C/F299CK160 641–643 aaa tgt F299 1058–1060 ctc tgt L161C/T300C L161 644–646 ctctgt T300 1061–1063 act tgt L289C/K294C L289 1028–1030 ctg tgt K2941043–1045 aag tgt αM Q163C/Q309C Q163 607–609 caa tgt Q309 1045–1047 cagtgt D294C/Q311C D294 1000–1002 gat tgt Q311 1051–1053 cag tgtQ163C/R313C Q163 607–609 caa tgt R313 1057–1059 cgg tgt αL; GenBankNM_002209 αM; GeneBank J03925

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, an isolated nucleic acid moleculeencoding a modified integrin I-domain polypeptide can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized.

The skilled artisan will further appreciate that further changes can beintroduced by mutation into the nucleotide sequence encoding a modifiedintegrin polypeptide, thereby leading to changes in the amino acidsequence of the encoded modified integrin polypeptide, without furtheraltering the structural characteristics or functional ability of themodified integrin polypeptide. For example, nucleotide substitutionsleading to amino acid substitutions at “non-essential” amino acidresidues can be made in the sequence encoding a modified integrinpolypeptide. A “non-essential” amino acid residue is a residue that canbe altered from the sequence of a modified integrin polypeptide withoutfurther altering the structure and/or biological activity. In accordancewith the methods of the invention, computational design and modeling areused to determine which amino acid residues are amenable to alterationin order to achieve the desired protein conformation.

Accordingly, the methods of the invention may include the use of nucleicacid molecules encoding modified integrin polypeptides that containchanges in amino acid residues that are not essential for activity.

Preferably, conservative amino acid substitutions are made at one ormore predicted non-essential amino acid residues. A “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a similar side chain. Families ofamino acid residues having similar side chains have been defined in theart. These families include amino acids with basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted nonessentialamino acid residue in a modified integrin polypeptide is preferablyreplaced with another amino acid residue from the same side chainfamily.

Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, for example,recombinant expression vectors, containing a nucleic acid encoding amodified integrin polypeptide (or a portion thereof), e.g., an integrinI-domain or I-like domain polypeptide or fusion protein. As used herein,the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the methods of the invention may include other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the-basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cells and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., modified integrinI-domain polypeptides, fusion proteins, and the like).

Accordingly, the invention provides a method for producing a modifiedintegrin polypeptide, e.g., a modified integrin I-domain polypeptide, byculturing ip. a suitable medium, a host cell of the invention (e.g., aprokaryotic or eukaryotic host cell) containing a recombinant expressionvector such that the protein is produced.

The recombinant expression vectors of the invention can be designed forexpression of modified integrin polypeptides or fusion proteins inprokaryotic or eukaryotic cells, e.g., for use in the methods of theinvention. For example, modified integrin I-domain polypeptides orfusion proteins can be expressed in bacterial cells such as E. coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility and/or stability ofthe recombinant protein; and 3) to aid in the purification of therecombinant protein by acting as a ligand in affinity purification.Often, in fusion expression vectors, a proteolytic cleavage site isintroduced at the junction of the fusion moiety and the recombinantprotein to enable separation of the recombinant protein from the fusionmoiety subsequent to purification of the fusion protein. Such enzymes,and their cognate recognition sequences, include Factor Xa, thrombin andenterokinase. Typical fusion expression vectors include pGEX (PharmaciaBiotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31–40), pMAL(New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway,N.J.) which fuse glutathione S-transferase (GST), maltose E bindingprotein, or protein A, respectively, to the target recombinant protein.Purified modified integrin I-domain fusion proteins (e.g., solubleI-domain-Ig) can be utilized to modulate integrin activity, as describedherein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301–315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60–89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21 (DE3) or HMS174(DE3) from a residentprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119–128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111–2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerevisiaeinclude pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229–234), pMFa(Kurjan and Herskowitz, (1982) Cell 30:933–943), pJRY88 (Schultz et al.,(1987) Gene 54:113–123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, modified integrin polypeptides can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol.3:2156–2165) and the pVL series (Lucklow and Summers (1989) Virology170:31–39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187–195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268–277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:235–275), in particular promoters of Tcell receptors (Winoto and Baltimore (1989) EMBO J. 8:729–733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729–740; Queen andBaltimore (1983) Cell 33:741–748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473–5477), endothelial cell-specific promoters (e.g., KDR/flkpromoter; U.S. Pat. No. 5,888,765), pancreas-specific promoters (Edlundet al. (1985) Science 230:912–916), and mammary gland-specific promoters(e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and EuropeanApplication Publication No. 264,166). Developmentally-regulatedpromoters are also encompassed, for example the murine hox promoters(Kessel and Gruss (1990) Science 249:374–379) and the α-fetoproteinpromoter (Campes and Tilghman (1989) Genes Dev. 3:537–546).

Another aspect of the invention pertains to host cells into which anucleic acid molecule encoding a modified integrin polypeptide of theinvention is introduced, e.g., a modified integrin I-domain nucleic acidmolecule within a recombinant expression vector or a modified integrinI-domain nucleic acid molecule containing sequences which allow it tohomologously recombine into a specific site of the host cell's genome.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, amodified integrin polypeptide or fusion protein can be expressed inbacterial cells such as E. coli, insect cells, yeast or mammalian cells(such as hematopoietic cells, leukocytes, K562 cells, 293T cells, humanumbilical vein endothelial cells (HUVEC), human microvascularendothelial cells (HMVEC), Chinese hamster ovary cells (CHO) or COScells). Other suitable host cells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acids encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a modified integrin polypeptide or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a modifiedintegrin polypeptide, e.g., a modified integrin I-domain polypeptide orfusion protein, for use in the methods of the invention. In oneembodiment, a host cell (into which a recombinant expression vectorencoding a modified integrin I-domain polypeptide or fusion protein hasbeen introduced) is cultured in a suitable medium such that a modifiedintegrin I-domain polypeptide or fusion protein is produced. In anotherembodiment, a modified integrin I-domain polypeptide or fusion proteinis isolated from the medium or the host cell. A recombinant cellexpressing a modified integrin polypeptide or fusion protein can also beadministered to a subject to modulate integrin activity.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into which amodified integrin I-domain polypeptide-coding sequences have beenintroduced. Such host cells can then be used to create non-humantransgenic animals in which exogenous modified integrin I-domainsequences have been introduced into their genome or homologousrecombinant animals in which endogenous integrin I-domain sequences havebeen altered. Such animals are useful for studying the function and/oractivity of a modified integrin I-domain molecule and for identifyingand/or evaluating modulators of modified integrin I-domain polypeptideactivity. As used herein, a “transgenic animal” is a non-human animal,preferably a mammal, more preferably a rodent such as a rat or mouse, inwhich one or more of the cells of the animal includes a transgene. Otherexamples of transgenic animals include non-human primates, sheep, dogs,cows, goats, chickens, amphibians, and the like. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal, thereby directing the expression of an encoded gene product inone or more cell types or tissues of the transgenic animal. As usedherein, a “homologous recombinant animal” is a non-human animal,preferably a mammal, more preferably a mouse, in which an endogenousintegrin I-domain gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducing amodified integrin I-domain-encoding nucleic acid into the male pronucleiof a fertilized oocyte, e.g., by microinjection, retroviral infection,and allowing the oocyte to develop in a pseudopregnant female fosteranimal. Intronic sequences and polyadenylation signals can also beincluded in the transgene to increase the efficiency of expression ofthe transgene. A tissue-specific regulatory sequence(s) can be operablylinked to a modified integrin I-domain transgene to direct expression ofa modified integrin I-domain protein to particular cells. Methods forgenerating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986).

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a modified integrin I-domain gene intowhich a deletion, addition or substitution has been introduced tothereby alter, e.g., functionally disrupt, the modified integrinI-domain gene. The modified integrin I-domain gene can be a human gene,but more preferably, is a non-human homologue of a human modifiedintegrin I-domain gene. For example, a mouse modified integrin I-domaingene can be used to construct a homologous recombination nucleic acidmolecule, e.g., a vector, suitable for altering an endogenous modifiedintegrin I-domain gene in the mouse genome. In a preferred embodiment,the homologous recombination nucleic acid molecule is designed suchthat, upon homologous recombination, the endogenous modified integrinI-domain gene is functionally disrupted (i.e., no longer encodes afunctional protein; also referred to as a “knock out” vector).Alternatively, the homologous recombination nucleic acid molecule can bedesigned such that, upon homologous recombination, the endogenousmodified integrin I-domain gene is mutated or otherwise altered butstill encodes functional protein (e.g., the upstream regulatory regioncan be altered to thereby alter the expression of the endogenousmodified integrin I-domain protein). In the homologous recombinationnucleic acid molecule, the altered portion of the modified integrinI-domain gene is flanked at its 5′ and 3′ ends by additional nucleicacid sequence of the modified integrin I-domain gene to allow forhomologous recombination to occur between the exogenous modifiedintegrin I-domain gene carried by the homologous recombination nucleicacid molecule and an endogenous modified integrin I-domain gene in acell, e.g., an embryonic stem cell. The additional flanking modifiedintegrin I-domain nucleic acid sequence is of sufficient length forsuccessful homologous recombination with the endogenous gene. Typically,several kilobases of flanking DNA (both at the 5′ and 3′ ends) areincluded in the homologous recombination nucleic acid molecule (see,e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced modified integrin I-domain gene has homologouslyrecombined with the endogenous modified integrin I-domain gene areselected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells can then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells:A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113–152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination nucleic acid molecules, e.g., vectors, or homologousrecombinant animals are described further in Bradley, A. (1991) CurrentOpinion in Biotechnology 2:823–829 and in PCT International PublicationNos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.;WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232–6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351–1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, antibodies, peptidomimetics, small molecules(organic or inorganic) or other drugs) which modulate integrin activity.These assays are designed to identify compounds, for example, that bindto an integrin I-domain polypeptide, e.g., an integrin I-domainpolypeptide in an active conformation, bind to other proteins thatinteract with an integrin I-domain polypeptide, induce binding, andmodulate the interaction of an integrin I-domain polypeptide with otherproteins, e.g., an integrin ligand, e.g., ICAM, and thus modulateintegrin activity.

As used herein, the term “modulator of integrin activity” includes acompound or agent that is capable of modulating or regulating at leastone integrin activity, as described herein. Modulators of integrinactivity may include, but are not limited to, small organic or inorganicmolecules, nucleic acid molecules, peptides, antibodies, and the like. Amodulator of integrin activity can be an inducer or inhibitor ofintegrin activity, e.g., cell adhesion or ligand binding. As usedherein, an “inducer of integrin activity” stimulates, enhances, and/ormimics an integrin activity. As used herein, an “inhibitor of integrinactivity” reduces, blocks or antagonizes an integrin activity.

As used interchangeably herein, an “integrin activity”, or an“integrin-mediated activity” refers to an activity exerted by anintegrin polypeptide or nucleic acid molecule on an integrin responsivecell, or on integrin ligand or receptor, as determined in vitro and invivo, according to standard techniques. In one embodiment, an integrinactivity is the ability to mediate cell adhesion events, e.g, cell tocell or cell to extracellular matrix adhesion. In another embodiment, anintegrin activity is the ability to transduce cellular signaling events.In yet another embodiment, an integrin activity is the ability to bind aligand, e.g., ICAM.

In a preferred embodiment, a soluble, recombinant high affinity integrinI-domain can be used to screen for small molecule antagonists thatinterfere with integrin ligand binding. Furthermore, antagonists, e.g.,antibodies, with direct/competitive and indirect/noncompetitive modes ofinhibition can be discriminated, based on comparison with effects onwild-type integrin I-domains which show minimal ligand binding activity.For example, an indirect inhibitor should inhibit ligand binding by anactivated, wild-type integrin I-domain, but not by a disulfide-lockedhigh affinity I-domain.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a modified integrin polypeptide on the cellsurface with a test compound and determining the ability of the testcompound to modulate (e.g., induce or inhibit) an integrin activity. Forexample, a cell expressing a modified integrin I-domain polypeptidestabilized in an open conformation on the cell surface is contacted witha test compound, and the ability of the test compound to modulateadhesion to an integrin ligand is determined, as described andexemplified herein.

In yet another embodiment, the ability of a test compound to modulateintegrin ligand binding can also be determined, for example, by couplinga modified integrin I-domain polypeptide that is stabilized in an openconformation with a detectable label such that the binding of themodified integrin polypeptide can be determined by detecting the amountof labeled integrin I-domain binding to an immobilized integrin ligand.

Animal-based model systems, such as an animal model of inflammation, maybe used, for example, as part of screening strategies designed toidentify compounds which are modulators of integrin activity. Thus, theanimal-based models may be used to identify drugs, pharmaceuticals,therapies and interventions which may be effective in modulatinginflammation and treating integrin-mediated disorders. For example,animal models may be exposed to a compound, suspected of exhibiting anability to modulate integrin activity, and the response of the animalsto the exposure may be monitored by assessing inflammatory activitybefore and after treatment. Transgenic animals, e.g., transgenic mice,which express modified integrin I-domain polypeptides as describedherein can also be used to identify drugs, pharmaceuticals, therapiesand interventions which may be effective in modulating inflammation andtreating integrin-mediated disorders

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulator ofintegrin activity can be identified using a cell-based assay, and theability of the agent to modulate integrin activity can be confirmed invivo, e.g., in an animal such as an animal model for inflammation.

Moreover, screening assays can be used to identify inducers of integrinactivity, for example, that mimic the activity of a integrinpolypeptide, e.g., the binding of an integrin to a ligand or receptor,or the activity of an integrin towards an integrin responsive cell. Suchcompounds may include, but are not limited to, peptides, antibodies, orsmall organic or inorganic compounds. In one embodiment, ananti-integrin antibody, e.g., an anti-LFA-1 antibody of the inventionwhich selectively binds to an open, activated conformer can be used toassess the ability of a test compound to activate integrin.

The test compounds can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412–421), or on beads (Lam (1991) Nature354:82–84), chips (Fodor (1993) Nature 364:555–556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865–1869) or on phage(Scott and Smith (1990) Science 249:386–390); (Devlin (1990) Science249:404–406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378–6382); (Felici (1991) J. Mol. Biol. 222:301–310); (Ladnersupra.).

This invention further pertains to novel agents identified by theabove-described screening assays. With regard to intervention, anytreatments which modulate integrin activity and/or inflammatory activityshould be considered as candidates for human therapeutic intervention.

Pharmaceutical Compositions

The nucleic acid molecules encoding modified integrin polypeptides,modified integrin polypeptides (e.g., modified I-domain polypeptides andfusion proteins), and active fragments thereof, anti-integrin I-domainantibodies, and integrin modulators (also referred to herein as “activecompounds”) DNA vaccines, or DNA vectors of the invention can beincorporated into pharmaceutical compositions suitable foradministration. As used herein, a “modulator” of integrin activity,e.g., inhibitors and inducers, includes a compound that modulates anintegrin activity, e.g., an integrin-mediated signaling event, anintegrin-mediated adhesion event, or integrin binding to a cognateligand. Integrin modulators include modified integrin I-domain or I-likedomain polypeptides of the invention, anti-integrin I-domainpolypeptides, as well as compounds identified in a screening assaydescribed herein. Such compositions typically comprise the compound,nucleic acid molecule, vector, protein, or antibody and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, ophthalmic, and rectal administration, including directinstallation into a disease site. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a soluble modified integrin I-domain fusion protein) inthe required amount in an appropriate solvent with one or a combinationof ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The administration of the active compounds of the invention may be foreither a prophylactic or therapeutic purpose. Accordingly, in oneembodiment, a “therapeutically effective dose” refers to that amount ofan active compound sufficient to result in a detectable change in thephysiology of a recipient patient. In one embodiment, a therapeuticallyeffective dose refers to an amount of an active compound sufficient toresult in modulation of an inflammatory and/or immune response. Inanother embodiment, a therapeutically effective dose refers to an amountof an active compound sufficient to result in the amelioration ofsymptoms of an inflammatory and/or immune system disorder. In anotherembodiment, a therapeutically effective dose refers to an amount of anactive compound sufficient to prevent an inflammatory and/or immunesystem response. In yet another embodiment, a therapeutically effectivedose refers to that amount of an active compound sufficient to modulatean integrin activity (e.g., a signaling activity, an adhesion activityor a ligand binding activity) as described herein.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of antibody,protein or polypeptide (i.e., an effective dosage) ranges from about0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight. Ranges intermediate to the aboverecited values, also are intended to be part of this invention. Forexample, ranges of span values using a combination of any of the aboverecited values as upper and/or lower limits are intended to be included.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to the severity of the disease or disorder, previous treatments,the general health and/or age of the subject, and other diseasespresent. Moreover, treatment of a subject with a therapeuticallyeffective amount of a protein, polypeptide, or antibody can include asingle treatment or, preferably, can include a series of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

In another preferred example, a subject is treated with an initialdosing of a therapeutically effective amount of an anti-integrinantibody, e.g., an anti-integrin antibody, e.g., an anti-LFA-1 antibody,which reacts with or binds to an I-domain of an integrin in the open oractive conformation, or an anti-integrin antibody, e.g., an anti-LFA-1antibody, which reacts with or binds to a modified LFA-1 I-domain,followed by a subsequent intermittent dosing of a therapeuticallyeffective amount of the antibody that is less than 100%, calculated on adaily basis, of the initial dosing of the antibody wherein the antibodyis administered not more than once per week during the subsequentdosing. In another embodiment, the subsequence dosing is two or moretimes per week. In another embodiment, the subsequence dosing is one ormore time every two weeks. In still another embodiment, the subsequencedosing is one or more times every three weeks. In yet anotherembodiment, the subsequence dosing is one or more times every fourweeks. In one embodiment, the subsequent dosing is less than about 50%,45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1%, calculated on a daily basis, of the initial dosing of theantibody. In one embodiment, the initial dosage is between 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. In a preferred embodiment, the initial dosage is lessthan 0.3 mg/kg body weight, e.g., between 0.001 to 0.30, e.g., 0.1,0.125, 0.15, 0.175, 0.2, 0.225, 0.25, and 0.275. Ranges intermediate tothe above recited values, also are intended to be part of thisinvention.

In yet another example, a subject is treated with an initial dosing of atherapeutically effective amount of an anti-integrin antibody, e.g., ananti-integrin antibody, e.g., an anti-LFA-1 antibody, which reacts withor binds to an I-domain of an integrin in the open or activeconformation, or an anti-integrin antibody, e.g., an anti-LFA-1antibody, which reacts with or binds to a modified LFA-1 I-domain,followed by a subsequent intermittent dosing of a therapeuticallyeffective amount of the antibody that is greater than 100%, calculatedon a daily basis, of the initial dosing of the antibody wherein theantibody is administered to the mammal not more than once per weekduring the subsequent dosing. In another embodiment, the subsequencedosing is two or more times per week. In another embodiment, thesubsequence dosing is one or more time every two weeks. In still anotherembodiment, the subsequence dosing is one or more times every threeweeks. In yet another embodiment, the subsequence dosing is one or moretimes every four weeks. In one embodiment, the initial dosage is between0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg bodyweight, more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight. In a preferred embodiment, theinitial dosage is less than 0.3 mg/kg body weight, e.g., between 0.001to 0.3, e.g., 0.1, 0.125, 0.15, 0.175, 0.2, 0.225, 0.25, and 0.275.Ranges intermediate to the above recited values, also are intended to bepart of this invention. Dosages for anti-integrin antibodies, e.g.,anti-LFA-1 are described in, for example, U.S. Pat. No. 5,622,700.

In still another example, an initial dosage is followed by the samedosage, for example, not more than once per week during the subsequentdosing. In another embodiment, the subsequence dosing is two or moretimes per week. In another embodiment, the subsequence dosing is one ormore time every two weeks. In still another embodiment, the subsequencedosing is one or more times every three weeks. In yet anotherembodiment, the subsequence dosing is one or more times every fourweeks.

Dosages for anti-integrin antibodies, e.g., anti-LFA-1 are described in,for example, U.S. Pat. No. 5,622,700.

In another embodiment, the an effective amount of an anti-inflammatoryor immunosuppressive agent to the mammal in combination with theantibody, either at the same time, or at different time points.

The present invention encompasses active agents which modulate anintegrin activity. An agent may, for example, be a small molecule. Forexample, such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

In certain embodiments of the invention, a modulator of integrinactivity is administered in combination with other agents (e.g., a smallmolecule), or in conjunction with another, complementary treatmentregime. For example, in one embodiment, an inhibitor of integrinactivity is used to treat an inflammatory or immune system disorder.Accordingly, the subject may be treated with an inhibitor of integrinactivity, and further treated with an anti-inflammatory orimmunosuppressive agent.

Further, an antibody, e.g., an anti-LFA-1 antibody, (or fragmentthereof) may be conjugated to a therapeutic moiety such as a cytotoxin,a therapeutic agent or a radioactive metal ion. The conjugates of theinvention can be used for modifying a given biological response, and thedrug moiety is not to be construed as limited to classical chemicaltherapeutic agents. For example, the drug moiety may be a protein orpolypeptide possessing a desired biological activity. Such proteins mayinclude, for example, a coagulation factor such as tissue factor; aprotein such as vascular endothelial growth factor (“VEGF”), plateletderived growth factor, and tissue plasminogen activator; biologicalresponse modifiers such as, for example, lymphokines, cytokines andgrowth factors; or a toxin.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243–56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2^(nd) Ed.), Robinson et al. (eds.), pp.623–53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers OfCytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (eds.), pp.475–506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303–16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119–58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The nucleic acid molecules of the invention, e.g., a nucleic acidmolecule encoding, for example, a high-affinity modified integrinI-domain polypeptide, or active fragment thereof, can be used as agene-based therapy alone, or, can be inserted into vectors and used asgene therapy vectors. Gene therapy is the insertion of a functioninggene into the cells of a patient (i) to correct an inborn error ofmetabolism, or (ii) to provide a new function in a cell (Kulver, K. W.,“Gene Therapy”, 1994, p. xii, Mary Ann Liebert, Inc., Publishers, NewYork, N.Y.). Vectors, e.g., viral vectors, may be used to introduce andstably express a gene normally expressed in mammals, for example, in alocation in the body where that gene is not naturally present. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054–3057). The gene therapy vector caninclude, for example, DNA encoding an antigen of interest to induce animmune response in the subject in vivo. Therefore, the modified integrinI-domain polypeptide, e.g., a high-affinity modified integrin I-domainpolypeptide, or active fragment thereof, acts as an adjuvant to producean increased antibody reaction to the antigen. The. pharmaceuticalpreparation of the gene therapy vector can include the gene therapyvector in an acceptable diluent, or can comprise a slow release matrixin which the gene delivery vehicle is imbedded. Alternatively, where thecomplete gene delivery vector can be produced intact from recombinantcells, e.g., retroviral vectors, the pharmaceutical preparation caninclude one or more cells which produce the gene delivery system.

The nucleic acid molecules of the invention can also be used in DNAvaccine formulations for therapeutic or prophylactic treatment ofintegrin-mediated disorders, e.g., inflammatory disorders. In oneembodiment, the DNA vaccine formulation comprises a nucleic acidmolecule encoding a modified integrin polypeptide, e.g., a modifiedintegrin I-domain polypeptide, or fragment thereof, coupled with anantigenic component, e.g., DNA encoding an antigenic component. As usedherein, an antigenic component is a moiety that is capable of binding toa specific antibody with sufficiently high affinity to form a detectableantigen-antibody complex. In another embodiment, the DNA vaccine furthercomprises a pharmaceutically acceptable carrier.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of an integrin-mediated disorderor having an integrin-mediated disorder such as an inflammatory orimmune disorder, and/or a cellular proliferative disorder. “Treatment”,as used herein, is defined as the application or administration of atherapeutic agent to a patient, or application or administration of atherapeutic agent to an isolated tissue or cell line from a patient, whohas a disease or disorder, a symptom of disease or disorder or apredisposition toward a disease or disorder, with the purpose of curing,healing, alleviating, relieving, altering, remedying, ameliorating,improving or affecting the disease or disorder, the symptoms of diseaseor disorder or the predisposition toward a disease or disorder. Atherapeutic agent includes, but is not limited to, nucleic acidmolecules, DNA vaccines, gene-based therapies, small molecules,peptides, antibodies, e.g., anti-LFA-1 antibodies, which react with orbind to modified I-domain polypeptides, ribozymes and antisenseoligonucleotides.

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the integrin I-domainpolypeptides of the present invention or modulators thereof according tothat individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject a disease or condition associated with a integrin-mediateddisorder by administering to the subject one or more integrin I-domainpolypeptides of the present invention or modulators thereof. Subjects atrisk for an integrin-mediated disorder can be identified by, forexample, any or a combination of diagnostic or prognostic assays asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the integrin-mediateddisorders, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofintegrin-mediated disorder, for example, appropriate integrin I-domainpolypeptides of the present invention, or modulators thereof, can beused for treating the subject. The appropriate agent can be determinedbased on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulatingexpression of integrin I-domain polypeptides or their activity fortherapeutic purposes (e.g., treating a subject at risk of anintegrin-mediated disorder or having an integrin-mediated disorder suchas an inflammatory or immune disorder, and/or a cellular proliferativedisorder). Accordingly, in an exemplary embodiment, the modulatorymethod of the invention involves contacting a cell with one or moreintegrin I-domain polypeptides of the present invention, or one or moremodulators thereof, e.g., an antibody which reacts of binds to anintegrin I-domain in an open conformation or a modified integrinI-domain polypeptide, e.g., an anti-LFA-1 antibody specific for an LFA-1I-domain in an open conformation or a modified LFA-1 I-domainpolypeptide. An agent that modulates integrin I-domain polypeptideactivity can be an agent as described herein, such as a nucleic acid ora protein, a target molecule of an integrin I-domain polypeptide (e.g.,a substrate), an antibody which reacts or binds to a modified integrinI-domain polypeptide, an integrin I-domain polypeptide agonist orantagonist, a peptidomimetic of an integrin I-domain polypeptide agonistor antagonist, or other small molecule. In one embodiment, the agentstimulates one or more integrin I-domain polypeptide activities.Examples of such stimulatory agents include active integrin I-domainpolypeptide protein and a nucleic acid molecule encoding integrinI-domain polypeptide that has been introduced into the cell. In anotherembodiment, the agent inhibits one or more integrin I-domain polypeptideactivities. Examples of such inhibitory agents include antisenseintegrin I-domain polypeptide nucleic acid molecules, gene therapyvectors, DNA vaccines, anti-integrin I-domain polypeptide antibodies,and integrin I-domain polypeptide inhibitors. These modulatory methodscan be performed in vitro (e.g., by culturing the cell with the agent)or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized associatedwith an integrin-mediated disorder. In one embodiment, the methodinvolves administering an agent (e.g., an agent identified by ascreening assay described herein), or combination of agents thatmodulates (e.g., upregulates or downregulates) integrin I-domainpolypeptide expression or activity.

3. Pharmacogenomics

The integrin I-domain polypeptide molecules of the present invention, aswell as agents, or modulators which have a stimulatory or inhibitoryeffect on integrin I-domain polypeptide activity (e.g., integrinI-domain polypeptide gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) an integrin-mediated disorder suchas an inflammatory or immune disorder, and/or a cellular proliferativedisorder. In conjunction with such treatment, pharmacogenomics (i.e.,the study of the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer anintegrin I-domain polypeptide molecule (and/or a modulator thereof) aswell as tailoring the dosage and/or therapeutic regimen of treatmentwith such molecule and/or modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10–11): 983–985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254–266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate aminopeptidase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000–100,000 polymorphic or variable sites on the humangenome, each of which has two variants). Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and the cytochrome P450enzymes CYP2D6 and CYP2C19) has provided an explanation as to why somepatients do not obtain the expected drug effects or show exaggerateddrug response and serious toxicity after taking the standard and safedose of a drug. These polymorphisms are expressed in two phenotypes inthe population, the extensive metabolizer (EM) and poor metabolizer(PM). The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., an integrinI-domain polypeptide molecule or integrin I-domain polypeptidemodulator) can give an indication whether gene pathways related totoxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with an integrinI-domain polypeptide molecule or modulator thereof, such as a modulatoridentified by one of the exemplary screening assays described herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures and sequence listing areincorporated herein by reference.

EXAMPLES Example 1 Design of LFA-1 and Mac-1 Mutants that are Locked inOpen or Closed Conformation

Current crystal and NMR structures of the LFA-1 I domain (Qu, A andLeahy, D J (1995) Proc Natl Acad Sci USA 92:10277–10281; Qu, A andLeahy, D J (1996) Structure 4:931–942; Kallen, J et al. (1999) J MolBiol 292:1–9) have a conformation that is similar to the low affinity,closed conformer of the Mac-1 I domain (1jlm) (Lee, J-O et al. (1995)Cell 80:631–638). Therefore, the high affinity, open conformer of theMac-1 I domain (1ido) (Lee, J-O et al. (1995) Structure 3:1333–1340) wasused to model a high affinity, open LFA-1 I domain. The template forthis model consisted of segments of the lido structure in regions wherethe Cα backbone differed significantly from the 1jlm structure, andsegments of the 1lfa structure in regions where 1ido and 1jlm weresimilar.

Briefly, I domains with the following protein data bank (PDB)identifiers were structurally superimposed using Cα carbons, the CDMALIGN algorithm of MODELLER 4 (Sali, A and Blundell, T L (1993) J MolBiol 234:779–815), and a gap extension penalty of 1 Å: Mac-1, 1ido and1jlm (Lee, J-O et al. (1995) Structure 3:1333–1340; Lee, J-O et al.(1995) Cell 80:631–638); LFA-1, 1lfa molecules A and B (Qu, A and Leahy,D J (1995) Proc Natl Acad Sci USA 92:10277–10281), 1zon and 1zop (Qu, Aand Leahy, D J (1996) Structure 4:931–942); and VLA-2, 1aox (Emsley, Jet al. (1997) J Biol Chem 272:28512–28517). The algorithm found 121framework residues that were utilized for superposition. A sequencealignment was then done. The 1ido and 1jlm structures were aligned bytheir sequence, and 1lfa molecule A and 1zon were aligned by structuralsimilarity to 1jlm. Using the structural superposition, and the sequencealignment, the distances between all Cα carbons at equivalent sequencepositions were calculated using a Microsoft Excel spreadsheet. This wasanalogous to the comparison between 1jlm and 1ido (Lee, J-O et al.(1995) Structure 3:1333–1340), except that LFA-1 I-domain structureswere included. For use as templates for the high affinity, open LFA-1 Idomain model, segments from 1lfa molecule A were chosen wheredifferences between all four I domains were small, or differencesbetween 1lfa and 1jlm (low affinity, closed LFA-1 and Mac-1 I domains)were greater than between 1ido and 1jlm (open and closed Mac-1 Idomains). Segments from 1ido were chosen when differences between 1idoand 1jlm were greater than between 1lfa and 1jlm. These segments werespliced together in regions where the backbones were as similar aspossible. Thus, the template utilized segments G128 to F136, M154 toL203, F209 to L234, T243 to I255, and E272 to A282 of 1lfa; and segmentsD140 to F156, G207 to T211, V238 to K245, R266 to R281, and R293 to K315of 1ido. No chain breaks were detected by LOOK™ (Molecular ApplicationGroup, Palo Alto, Calif.) in the spliced template, dubbed lfa-mac.Models of a high affinity open form of LFA-1 were made with MODELLER 4™using this template, the Mg²⁺ and water molecules 403 and 404 of 1ido,with heteroatom, water, and hydrogen input turned on, and dynamicColoumb turned on. The resulting model (lfa_hi.063) followed thetemplate Cα coordinates closely (RMS=0.12 Å). The QUACHK score (Vriend,G (1990) J Mol Graph 8:52–56) is excellent (−0.135 compared to −0.215for the lfa-mac template, −0.08 for 1ido, and 0.0 for 1lfa).

The SSBOND program (Hazes, B and Dijkstra, B W (1988) ProteinEngineering 2:119–125) was used to identify positions where disulfidebonds could be introduced by mutating two appropriately positioned pairsof residues to cysteine. It was hypothesized that it might be possibleto use disulfide bonds to trap the LFA-1 I domain in either the open orclosed conformations.

The high affinity open LFA-1 I domain model (the 1fa_hi.063 model wasexamined and two low affinity closed LFA-1 I domain structures, 1lfa and1zon, with SSBOND and found 14 to 19 pairs of such residues in eachstructure. Out of these, one pair of residues in the high affinity openmodel, and one pair of residues in the low affinity closed structures,underwent large movements between the two conformers, such thatdisulfide bond formation could only occur in one conformer (FIG. 1).These disulfides bridge β-strand 6 to the C-terminal α-helix, α6. Thenumbering of β-strands and α-helices differs among I domains; we use auniform nomenclature (Huang, C et al. (2000) J Biol Chem, 275:21514–24).Helix α6 moves 10 Å along its axis down the body of the I domain in thehigh affinity open structure, and this movement is accompanied by acomplete remodeling and downward shift of the loop between β6 and α6.Cysteines introduced in place of K287 and K294 were predicted to form adisulfide only in the high affinity open conformer, and thus lock the Idomain in the high affinity open state (FIG. 2). The Cβ carbons of K287and K294 are predicted to be 3.8 Å apart in the high affinity open model(1fa_hi.063), within the range of 3.41 to 4.25 Å that is ideal fordisulfide formation, and after checking for Cβ-Sγ and Sγ-Sγ distances,were found to have four favorable sidechain-disulfide conformations. Bycontrast, in the low affinity closed conformers 1lfa and 1zon, the Cβatoms of these residues are 8.9 to 9.2 Å apart (FIG. 2).

Cysteines introduced in place of L289 and K294 were predicted to form adisulfide only in the low affinity closed conformer (FIG. 2), and thuslock the I domain in the low affinity closed state. The Cβ carbons ofL289 and K294 are 3.9 to 4.0 Å apart in the low affinity closed 1lfa and1zon conformers, within the favorable range, although favorable cysteinesidechain conformations were not found. Nonetheless, the α-helix inwhich residue 294 is present shows small displacements between 1lfa,1zon, and the recent NMR structure (Qu, A and Leahy, D J (1995) ProcNatl Acad Sci USA 92:10277–10281; Qu, A and Leahy, D J (1996) Structure4:931–942; Kallen, J et al. (1999) J Mol Biol 292:1–9), and it wasexpected that a disulfide could form with minor adjustment of theα-helix. By contrast, in the high affinity open model, the Cβ atoms ofthese residues are predicted to be 8.0 Å apart (FIG. 2).

Models were also built in which the predicted cysteines were present anddisulfide bonds were formed if appropriate using the PATCH DISULFIDEroutine of MODELLER 4 (FIG. 2); however, it should be noted that all Cβatom distances reported here are based on models or structures withoutintroduced disulfides.

In addition to the computational search for pairs of cysteinesubstitutions to form conformation-specific disulfide bridge, thestructure-oriented manual approach (or visual inspection) was also used.Regions of I domains that differ in conformation between the open andclosed conformations were inspected for positions in which pairs ofcysteines could be introduced that would form disulfides that wouldfavor one conformation over the other. Thus, the region of theconformationally mobile C-terminal α-helix and the preceding loop wereexamined for positions in which one cysteine could be introduced, andstructurally adjacent regions were searched for positions where a secondcysteine could be introduced that would form a disulfide bond. Pairs ofresidues whose side-chains face towards one another were chosen. Thedistance between the Cα and Cβ atoms of each of these pairs was measuredby software Look™ both in the open and closed conformation. The idealseparation for cysteine Cβ carbons for formation of a disulfide bond isreported to be 3.41 to 4.25 Å. However, the crystal structures or modelsfrom which these were measured represent average positions of snapshots,whereas proteins are dynamic and exhibit atomic mobility. Furthermore,structural adjustments are possible to accommodate disulfide bonds. Muchmore adjustment is expected to be possible in loops and a-helices thanin β-sheets. Therefore greater distances were predicted to be allowablefor disulfide formation when one of the residues was in a loop or helix.

For αL, 4 pairs of cysteine substitutions were found where the Cα-Cα andCβ-Cβ distances were more favorable for disulfide formation in the openconformation than in the closed conformation; E284C/E301C, L161C/F299C,K160C/F299C, and L161C/T300C (Table 1).

For αM, 4 pairs of cysteine substitutions were found where the Cα-Cα andCβ-Cβ distances were more favorable for disulfide formation in the openconformation than in the closed conformation: Q163C/Q309C, Q298C/N301C,D294C/T307C, and D294C/Q311c (Table 7), and one pair of cysteinesubstitutions where the Cα-Cα and Cβ-Cβ distances were more favorablefor disulfide formation in the closed conformation than in the openconformation: Q163C/R313C. Additionally, F297C/A304C, which is ananalogous mutation to K287C/K294C in αL, was included.

Example 2 Construction and Expression of LFA-1 Cysteine SubstitutionMutants

Five open αL I-domain mutants were generated. To generate the highaffinity open mutant K287C/K294C, the K287 and K294 in the I-domain ofthe αL subunit were replaced by cysteines. To generate the high affinityopen mutant E284C/E301C, the E284 and E301 in the I-domain of the αLsubunit were replaced by cysteines. In addition, threeintermediate-affinity open αL I-domain mutants were made, and areidentified herein as follows: L161C/F299C, K160C/F299C, and L161C/T300C.L161 C/F299C was made by substituting cysteines for the L161 and F299.K160C/F299C was made by substituting cysteines for the K160 and F299.L161C/T300C was made by substituting cysteines for the L161 and T300.The low affinity closed mutant L289C/K294C was made by substitutingcysteines for the L289 and K294. The distance between mutated residuesfor these six mutant is shown in Table 1, below. Also, single cysteinesubstitution mutants K287C, L289C and K294C were generated.

TABLE 1 Cα and Cβ between mutated residues in either open or closedconfirmation open closed conformation conformation Cα Cβ Cα Cβ αLI-domain (A) (A) (A) (A) Locked open K287C/K294C 6.32 3.75 10.72 9.08E284C/E301C 9.12 6.96 12.88 12.52 L161C/F299C 9.16 8.09 11.87 11.38K160C/F299C 9.97 7.75 9.83 7.96 L161C/T300C 12.30 13.00 13.50 14.87Locked closed L289C/K294C 7.90 7.96 6.19 3.86 The distance betweenwild-type residues was measured by Look ™ software in open conformation(lfa_hi.063) or closed conformation (1lfaA).

The human αL cDNA was contained in vector AprM8, a derivative of CDM8(Seed, B and Aruffo, A (1987) Proc Natl Acad Sci USA 84:3365–3369).Overlap extension PCR was used to generate cysteine substitutionmutations in the αL I-domain (Ho, S N et al (1989) Gene 77:51–59;Horton, R M et al. (1990) BioTechniques 8:528). The outer left primerfor PCR extension was complementary to the vector sequence at 5′ to theEcoRI site at position 1826, and the outer right primer was 3′ to theEcoRI site in the αL cDNA. The inner primers were designed for eachindividual mutation and contained overlapping sequences. Wild-type αLCDNA in AprM8 was used as template for the first PCR reaction. Thesecond PCR product was digested with EcoRI and ligated into the samesite in the wild-type αL cDNA in AprM8. The correct orientation of theinsert was confirmed by restriction enzyme digestion. All mutations wereconfirmed by DNA sequencing.

For stable expression, the XbaI fragment of αL wild-type and mutant cDNAwas subcloned into the same site of the stable expression vector pEFpuro(Lu, C and Springer, T A. (1997) J Immunol 159:268–278).

The mutated αL subunit was transiently coexpressed with the β2 subunitin 293T cells, and cell surface expression of the αL/β2 complex wasdetermined by flow cytometry with monoclonal antibody TS2/4 to the αLsubunit in the αL/β2 complex.

Briefly, human embryonic kidney 293T cells (SV40 transformed) werecultured in DMEM medium supplemented with 10% fetal bovine serum (FBS),2 mM glutamine and 50 μg/ml gentamycin. 293T cells were transientlytransfected using the calcium phosphate method (DuBridge, R B et al.(1987) Mol Cell Biol 7:379–387; Heinzel, S S et al. (1988) J Virol62:3738–3746). Briefly, 7.5 μg of wild-type or mutant αL cDNA in plasmidAprM8 and 7.5 μg of β2 cDNA in AprM8 were used to co-transfect one 6-cmplate of 70–80% confluent cells. Two days after transfection, cells weredetached from the plate with Hanks' balanced salt solution (HBSS)containing 5 mM EDTA for LFA-1 expression and functional analyses.

Flow cytometric analysis was performed as previously described (Lu, Cand Springer, T A (1997) J Immunol 159:268–278). Briefly, cells werewashed and resuspended in L15 medium (Sigma) supplemented with 2.5% FBS(L15/FBS). 1×10×10⁵ cells were incubated with primary antibodies in 100μL15/FBS on ice for 30 min. Monoclonal antibodies were used at finalconcentration of 1:20 hybridoma supernatant, 1:200 ascites, or 10 μg/mlpurified IgG. Cells were then washed twice with L15/FBS, and incubatedwith FITC-conjugated goat anti-mouse IgG (heavy and light chain, ZymedLaboratories, San Francisco, Calif.) for 30 min on ice. After washing,cells were resuspended in cold PBS and analyzed on a FACScan (BectonDickinson, San Jose, Calif.).

As shown in FIG. 3A, the predicted high and low affinity mutants, andthe single cysteine substitution mutants expressed similar levels ofcell surface αL/β2 complex.

To test whether introducing the cysteines affected the overallconformation of the I-domain, a panel of monoclonal antibodies todifferent regions in the I-domain were tested for their reactivity withthe I-domain mutants. The monoclonal antibodies used in these studiesare as follows:

The mouse anti-human αL (CD11a) monoclonal antibodies TS1/11, TS1/12,TS1/22, TS2/4, TS2/6 and TS2/14; anti-β2 (CD18) monoclonal antibodiesTS1/18, CBRLFA-1/2, and CBRLFA-1/7; mAb YFC51; and the nonbinding mAbX63 have been described previously (Sanchez-Madrid, F et al. (1982) ProcNatl Acad Sci USA 79:7489–7493; Hale, L P et al. (1989) Arthritis Rheum32:22–30; Petruzzelli, L et al. (1995) J Immunol 155:854–866).Monoclonal antibodies BL5, F8.8, 25-3-1, May.035, CBRLFA-1/9,CBRLFA-1/1, S6F, and May.017 were described in Leukocyte Type V and wereobtained from the Fifth International Leukocyte Workshops.

Monoclonal antibodies X63 and TS1/11 were used as hybridoma supernatantsat a 1:20 dilution; monoclonal antibodies TS1/12, DBRLFA-1/2, CBRLFA-1/7and YFC51 were used as purified IgG at 10 μg/ml; monoclonal antibodiesTS1/2, TS2/14, TS 1/18 and TS2/4 used as ascites at a 1:200 dilution;and all monoclonal antibodies from the Fifth International LeukocyteWorkshops were used at a 1:200 dilution. All of the antibodies, exceptfor CBRLFA-1/1, bound to the mutants K287C/K294C and L289C/K294C andwild-type LFA-1 equally well (Table 2), indicating that the cysteinesubstitutions did not disrupt the I-domain structure. Binding ofmonoclonal antibody CBRLFA-1/1 to the high-affinity open mutantK287C/K294C was reduced to 40–50% of wild-type, however, this antibodyreacted with mutant L289C/K294C and the single cysteine substitutionmutants K287C, L289C and K294C as well as wild-type. Since antibodyCBRLFA-1/1 maps to residues 301–359 (Huang, C and Springer, T A (1995) JBiol Chem 270:19008–19016), and single Cys substitution for K287 andK294 did not affect binding of this antibody, it is likely that reducedbinding of CBRLFA-1/1 to mutant K287C/K294C was an indirect effect.Therefore, the conformation at the interface between the I- andβ-propeller domains in mutant K287C/K294C may be different from that inwild-type LFA-1.

The reactivity of antibody to the β-propeller domain of αL and to the β2subunit with mutants K287C/K294C and L289C/K294C was similar to that ofwild-type LFA-1, confirming that the structure of other domains of LFA-1molecule was not affected by the mutations.

TABLE 2 Reactivity of antibodies with LFA-1 cysteine substitutionmutants (% wild-type binding) K287C/K294C L298C/K294C K287C L289C K294CMab epitope 293T K562 293T K562 293T 293T 293T I-domain BL5 119–153,185–215  92.4 ± 11.29 92.39 85.79 ± 16.4  97.61 93.35 92.44 88.31 F8.8119–153, 185–215 93.70 102.15  83.56 93.88 95.86 99.63 95.47 CBRLFA-1/9119–153, 185–215 ND 84.7  ND ND ND ND ND TS2/6 154–183 84.88 ± 5.64 89.24 78.59 ± 2.62  95.89 91.39 88.24 91.67 May.035 185–215 92.61 ± 8.4 92.59 82.14 ± 14.15 101.10  95.8  95.4  106.39  TS1/11 185–215 94.3695.96 93.67 104.54  ND ND ND TS1/12 185–215 88.66 87.32 101.98  105.63 99.32 103.89  93.68 TS1/22 185–302 95.85 ± 12.04 93.06 90.96 ± 8.11 110.49  102.99  96.21 92.24 TS2/14 250–303 85.54 ± 9.38  95.41 83.31 ±10.59 102.85  102.6  100.4  102.83  25-3-1 250–303 93.06 88.48 90.93 85.66 ND ND ND CBRLFA-1/1 I- and β-propeller 43.59 ± 0.58  55.53 95.89± 7.74  118.44 86.11 93.32 89.41 S6F1 β-propeller 89.39 97.38 95.32 85.69 98.3  86.39 92.34 β2 subunit TS1/18 I-like domain 99.82 ± 10.4797.42 95.72 ± 4.67  105.71  87.88 87.35 107.58  YFC51 I-like domain102.63  100.73  95.09 110.96  ND ND ND CLBLFA-1/1 I-like domain ND 96.48ND 100.50  ND ND ND CBRLFA-1/7 C-terminal region 95.32 95.25 91.68 97.19ND ND ND Wild-type LFA-1 and LFA-1 mutant K287C/K294C, L289C/K294C,K287C, L289C, and K294C were transiently expressed on the surface of293T cells or stably expressed on K562 transfectants. Reactivity ofantibodies with the transfectants was determined by flow cytometry. Meanfluorescence of each antibody binding was normalized to the meanfluorescence of mAb TS2/4 binding, except for CBRLFA-1/9 that wasnormalized to mAb TS1/22 binding. TS2/4 bound to wild-type LFA-1 and themutants equally well. The results are expressed as percent of wild-typebinding. Data are mean ± SD of at least two independent FCASexperiments. For some antibodies, only one experiment was done. ND: notdetermined.

Example 3 Ligand Binding Activity of LFA-1 Cysteine Substitution Mutants

The ability of the LFA-1 cysteine substitution mutants to bind to theLFA-1 ligand ICAM-1 was determined. 293T cell transfectants that expresswild-type LFA-1 and the predicted high-affinity open I-domain mutantK287C/K294C showed constitutively strong binding to immobilized ICAM-1(FIG. 4A). By contrast, the low-affinity closed mutant L289C/K294C didnot bind to ICAM-1. Whereas the single cysteine substitution mutantsK287C and L289C exhibited reduced binding to ICAM-1, binding of mutantK294C was comparable to that of the wild-type. Binding of mutants K287Cand L289C was increased by the activating monoclonal antibody CBRLFA-1/2to a level similar to wild-type binding. However, CBRLFA-1/2 was notable to activate binding of the low-affinity closed mutant L289C/K294Cto ICAM-1 (FIG. 4A). Similar results were obtained with two other LFA-1activating monoclonal antibodies Kim127 and Kim185. To further study thefunction of the predicted high affinity mutant K287C/K294C and lowaffinity closed mutant L289C/K294C, stable K562 transfectants thatexpress these mutants were generated.

Briefly, the human erythroleukemia cell line K562 was cultured in RPMI1640, 10% FBS and 50 μg/ml gentamycin. For generating stable K562 celllines, 2 μg of PvuI-linearized pEFpuro containing αL subunit cDNA wascotransfected with 40 μg of SfiI-linearized AprM8 containing the β2subunit cDNA by electroporation at 250V and 960 μF. Transfectants wereselected for resistance to 4 μg/ml puromycin (Sigma), and subcloned bylimiting dilution. All stable cell lines were maintained in RPMI 1640,10% FBS supplemented with 4 μg/ml puromycin.

Clones of the transfectants that expressed similar levels of cellsurface LFA-1, as determined by flow cytometry using monoclonal antibodyTS2/4 (FIG. 3B), were tested for their ability to bind to immobilizedICAM-1, as previously described (Lu, C and Springer, T A (1997) JImmunol 159:268–278).

Briefly, ICAM-1 was purified from human tonsil, and coated to 96-wellplates as described previously (Lu, C and Springer, T A (1997) J Immunol159:268–278). Cells were labeled with a florescence dye2′,7′-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein, acetoxymethylester (BCECF-AM), and resuspended to 1×10⁶/ml in L15/FBS. 50 μl cellsuspension was mixed in ICAM-1 coated wells with an equal volume ofL15I/FBS in the absence or presence of monoclonal antibody (CBRLFA-1/2,10 μg/ml). Monoclonal antibodies were used at final concentration of1:20 hybridoma supernatant, 1:200 ascites, or 10 μg/ml purified IgG. Fortesting the effect of divalent cations, BCECF-AM-labeled cells werewashed 2× with TS buffer, pH7.5 (20 mM Tris, pH 7.5, 150 mM NaCl)containing 5 mM EDTA, followed by 2 washes with TS buffer, pH7.5. Cellswere then resuspended to 5×10⁵/ml in the TS buffer, pH7.5 supplementedwith 1 mM MgCl₂/CaCl₂, MgCl₂, MnCl₂ or 5 mM EDTA, and 100 μl cellsuspension was added to ICAM-1 coated wells. After incubation at 37° C.for 30 minutes, unbound cells were washed off on a Microplate Autowasher(Bio-Tek Instruments, Winooski, Vt.). The fluorescence content of totalinput cells and the bound cells in each well was quantitated on aFluorescent Concentration Analyzer (IDEXX, Westbrook, Me.). The boundcells were expressed as a percentage of total input cells per samplewell.

K562 transfectants that express wild-type LFA-1 showed low basal bindingto ICAM-1, and binding was greatly increased by the activatingmonoclonal antibody CBRLFA-1/2 (FIG. 4B). By contrast, cells expressingthe predicted high-affinity open mutant K287C/K294C strongly bound toICAM-1, and monoclonal antibody CBRLFA-1/2 did not further enhancebinding of this mutant, whereas the predicted low-affinity closed mutantL289C/K294C did not binding to ICAM-1 even in the presence of theactivating antibody.

The effect of divalent cations on binding of K562 transfectants toICAM-1 was also examined. As shown in FIG. 4C, binding of mutantK287C/K294C to ICAM-1 was abolished in the presence of EDTA, confirmingthat ligand binding of mutant K287C/K294C is divalent cation dependent.Whereas binding of wild-type LFA-1 was greatly enhanced by Mn²⁺, and toa lesser degree by Mg²⁺, the presence of Mn²⁺ and Mg²⁺ did not increasebinding of the low-affinity closed mutant L289C/K294C to ligand.

The binding of soluble ICAM-1 to K562 transfectants that expressedwild-type LFA-1, mutant K287C/K294C, or mutant L289C/K294C was alsoassessed. Briefly, a soluble ICAM-1-IgA chimera containing the 5 Igdomains of human ICAM-1 was purified from the culture supernatant ofstable CHO transfectants by monoclonal antibody R6.5 affinitychromatography as previously described (Martin, S et al. (1993) J Virol67:3561–3568). K562 transfectants were washed once with L15/FBS, andresuspended in the same buffer to 1×10⁷/ml. 25 μl cell suspension wasmixed with 25 μl L15/FBS containing ICAM-1-IgA fusion protein at finalconcentration 100 μg/ml in the presence or absence of antibodyCBRLFA-1/2 (10 μg/ml), and incubated at 37° C. for 30 minutes. Afterincubation, cells were washed once in L15/FBS, and incubated withFITC-conjugated anti-human IgA (Sigma) at room temperature for 20minutes. After 2 washes, cells were resuspended in PBS, and analyzed ona FACScan (Becton Dickinson, San Joe, Calif.).

As shown in FIG. 5, the soluble ICAM-1-IgA fusion protein bound to cellsexpressing the high-affinity open mutant K287C/K294C, and binding wasfurther increased in the presence of the activating monoclonal antibodyCBRLFA-1/2. However, the ICAM-1 fusion protein did not bind to thetransfectants that expressed wild-type LFA-1 or the low affinity closedmutant L289C/K294C in the absence or presence of monoclonal antibodyCBRLFA-1/2, and binding was not detected at a higher ICAM-1 fusionprotein concentration (300 μg/ml).

Taken together these data indicate that the high affinity open mutantK287C/K294C is constitutively active, whereas the low-affinity closedmutant L289C/K294C appears to be locked in an inactive state and lacksligand binding ability.

In another study, a panel of monoclonal antibodies to different domainsof the αL and β2 subunits were tested for their inhibitory effect onligand binding of wild-type LFA-1 and mutant K287C/K294C. The resultsobtained with the 293T transient transfectants and K562 stabletransfectants were similar, and summarized in Table 3. Although allantibodies, except for CBRLFA-1/1, reacted with the high affinity openmutant K287C/K294C as well as wild-type (Table 2), they showeddifferential inhibition on ligand binding of wild-type LFA-1 and mutantK287C/K294C.

As shown in Table 3, the I-domain antibodies differentially inhibitedbinding of wild-type LFA-1 and the high affinity open mutant K287C/K294Cto ICAM-1. Monoclonal antibodies BL5, F8.8, CBRLFA-1/9, May.035, TS1/22and TS2/6 strongly inhibited binding of both wild-type and mutantK287C/K294C, and the levels of inhibition to wild-type LFA-1 and themutant were similar. While monoclonal antibodies TS1/11 and TS1/12inhibited >90% binding of transfectants that express wild-type LFA-1,these antibodies showed reduced inhibition on binding of mutantK287C/K294C (40–60%). Monoclonal antibodies TS2/14, 25-3-1 andCBRLFA-1/1 that showed >90% inhibition on binding of wild-type had no tolittle inhibition on mutant K287C/K294C binding to ICAM-1. While theβ-propeller domain antibody S6F1 and TS2/4 and antibody CBRLFA-1/7 tothe C-terminal region of the β2 subunit did not inhibit binding of bothwild-type and mutant K287C/K294C, all five antibodies to the β2conserved domain, TS1/18, YFC51, CLBLFA-1/1, May.017, and 6.5E,inhibited binding of wild-type LFA-1 (>90% inhibition), but did notinhibit binding of mutant K287C/K294C.

Antibodies to the β-propeller domain and to the C-terminal region of β2did not inhibit binding of wild-type LFA-1, or mutant K287C/K294C.Antibodies to the I-like domain of the β subunit blocked binding ofwild-type LFA-1 to ICAM-1, but did not block mutant K287C/K294C.

TABLE 3 Differential inhibition of antibodies on binding of wild-typeLFA-1 and mutant K287C/K294C to immobilized ICAM-1 % in hibitionwild-type LFA-1 K562 K287C/K294C MAb epitope 293T (+CBRLFA-1/2) 293TK562 RR1/1 I-CAM-1 95.98 ND 97.89 ND I-domain BL5 119–153, 185–215 97.01± 1.63 97.54 91.06 ± 3.8  90.68 ± 6.23 F8.8 119–153, 185–215 94.51 97.6191.94 98.18 CBRLFA-1/9 119–153, 185–215 ND 97.83 ND  3.60 TS2/6 154–18396.84 ± 1.73 91.76 ± 4.67  79.09 ± 10.06 88.12 ± 7.40 May.035 185–21596.20 ± 0.57 95.80 ± 1.66 97.43 ± 1.52 93.33 ± 2.54 TS1/11 185–215 94.1296.55 45.18 41.30 TS1/12 185–215 95.68 ± 3.92 97.46 ± 0.66 48.96 ± 9.5263.67 ± 8.13 TS1/22 250–303 95.77 96.94 ± 0.79 95.07 93.56 ± 4.79 TS2/14250–303 94.47 ± 2.34 96.24 ± 1.70  2.95 ± 9.87  8.55 ± 0.66 25-3-1250–303 90.49 92.01 ± 0.36  3.71  2.53 ± 4.10 CBRLFA-1/1 I- andβ-propeller 92.52 ± 1.68 94.69 ± 5.22  9.03  2.85 ± 4.90 S6F1β-propeller ND  6.19 ND  9.70 TS2/4 β-propeller ND  6.99 ND  2.82 β2subunit TS1/18 I-like domain ND 98.48 ND  5.90 YFC51 I-like domain ND98.43 ND  0.08 CLBLFA-1/1 I-like domain ND 94.63 ND  6.69 May.017 I-likedomain ND 97.76 ND  2.98 6.5E I-like domain ND 98.36 ND  5.79 CBRLFA-1/7C-terminal region ND  5.04 ND  5.77 Wild-type LFA-1 and LFA-1 mutantK287C/K294C were transiently expressed on the surface of 293T cells orstably expressed in K562 transfectants. Binding of the transfectants toimmobilized ICAM-1 was determined in the presence of the indicatedantibodies. For binding of K562 transfectants that express wild-typeLFA-1, the cells were preincubated with the activating mAb CBRLFA-1/2 at10 μg/ml for 30 min. Data shown are % inhibition ± SD of at least twoindependent experiments. % inhibition is defined as % bound cells in thepresence of the indicated mAb/% bound cells in the presence of thenonbinding mAb X63 × 100. For some antibodies, only one experiment wasdone. However, in each experiment, each antibody was repeated intriplicate, and the standard deviation of the triplicate samples was<5%. ND: not determined.

Taken together, these results suggest that a subset of I-domainantibodies and antibodies to the β2 conserved domain do not directlyblock LFA-1 binding to ICAM-1, and that the high-affinity open mutantK287C/K294C appears to be conformationally locked in a high affinityopen state, and thus, antibodies that block ligand binding via indirectmechanisms could not block binding of mutant K287C/K294C to ICAM-1.

The high affinity open I-domains of the invention can be used todiscriminate between direct/competitive and indirect/non-competitivemodes of inhibition of LFA-1. For example, the LFA-1 inhibitorlovastatin binds to the I-domain in a hydrophobic pocket formed by the βsheet and the C-terminal α-helix (Kallen, J et al. (1999) J Mol Biol292:1–9) and thus inhibits LFA-1 by an indirect mechanism. Accordingly,the ability of lovastatin to inhibit ligand binding of the high-affinityI-domain (K287C/K294C) was assessed. Lovastatin dissolved in DMSO at 50mM was diluted in assay buffer. Cells (10⁶/ml) labeled with BCECF-AMwere preincubated with lovastatin (0–50 μM) at 37° C. for 15 minutes,then transferred to a 96 well plate coated with ICAM-1 and furtherincubated at 37° C. for 30 minutes in the presence or absence ofactivating monoclonal antibody (CBR LFA1/2) or MnCl₂. L15 mediumsupplemented with fetal bovine serum (L15/FBS) which contains Ca2+ andMg2+ was used for wild-type αLβ2 activated by antibody CBR LFA1/2. and20 mM HEPES pH7.4, 140 mM NaCl, 1 mM MnCl2, 2 mg/ml glucose, 1% BSA wasused for activation by Mn2+.

As shown in FIG. 6, lovastatin inhibits ICAM-1 binding by cellsexpressing wild-type LFA-1 and stimulated with Mn²⁺ or antibody(CBRLFA1/2), but does not interfere with ligand binding by the highaffinity open K287C/K294C mutant (HA/aLb2).

Example 4 Expression and Function of Isolated Wild-Type and MutantLFA-1I-Domains

To further examine the function of the predicted high and low affinitymutants, the wild-type I-domain and the I-domains of mutant K287C/K294Cand L289C/K294C from residues V130 to A338 were expressed on the surfaceof K562 cells by the transmembrane domain of the PDGF receptor.

To construct the isolated, cell-surface expressed I domains, DNAsequences that encode the signal peptide and the following 6 amino acidsfrom the 5′ end of repeat II of αL were ligated to the sequencesencoding residues V130-A338 that contains the I domain. HindIII and SalIsites were introduced immediately adjacent to the 5′ and 3′ ends of thisfragment, respectively. The HindIII-SalI fragment was subcloned in frameat the 5′ to the c-myc tag and the PDGF receptor (PDGFR) transmembranedomain in vector pDisplay™ (Invitrogen), and further subcloned intopcDNA3.1/Hygro using HindIII and NotI. All DNA amplification was carriedout with Pfu DNA polymerase (Stratagene), and the final constructs wereverified by DNA sequencing.

For generating stable K562 transfectants that express the I-domain onthe surface, 20 μg of SspI-linearized pcDNA3.1/Hygro(+) containing thesequences encoding the I domain and the PDGFR transmembrane domain wasused to transfect K562 cells by electroporation as described above.Transfectants were selected for resistance to 100 μg/ml hygromycin B,and were further subcloned by cell sorting and limiting dilution; clonesthat expressed similar levels of surface wild-type and mutant Idomain-PDGFR were selected for functional studies. Stable cell lineswere maintained in RPMI medium 1640 supplemented with 10% FBS and 100μg/ml hygromycin B. Cell surface expression of the isolated I-domainswas determined by flow cytometry using antibody TS1/22 to the I-domain(FIG. 7). Two clones from each transfectant were selected and tested forbinding to immobilized ICAM-1, and similar results were obtained witheach of the two clones (FIG. 8A). Transfectants that expressed intactwild-type LFA-1 showed low basal binding to ICAM-1. However, cells thatexpressed the isolated wild-type I-domain and the mutant L289C/K294CI-domain did not bind to ICAM-1. This suggests that the isolatedwild-type I-domain alone is not sufficient to mediate strong and stableinteraction with ligand (Knorr, R and Dustin, M L (1997) J Exp Med186:719–730). By contrast, cells that expressed the mutant K287C/K294CI-domain showed strong binding to ICAM-1.

If the constitutive ligand binding activity of mutant K287C/K294C is dueto the formation of a disulfide bond between the introduced C287 andC294, disruption of the disulfide bond with a reducing agent wouldabolish ligand binding ability of the mutant. Accordingly, thetransfectants were treated with the reducing agent DTT (10 mM) inL15/FBS containing Mg²⁺ and Ca²⁺, and the ability of transfectants tobind to ICAM-1 was assessed. As shown in FIG. 8A, binding of the cellsurface-expressed mutant K287C/K294C I-domain to ICAM-1 who abolishedafter DTT treatment. By contrast, DTT increased binding of intactwild-type LFA-1, and similar results were observed with intact αIIbβ3integrin. DTT treatment presumably disrupts disulfide bonds in theintact molecule that constrain the integrin in an inactive conformation.However, DTT treatment did not affect binding of the isolated wild-typeI-domain or the mutant L289C/K294C I-domain. Since there is no other nodisulfide bond in the LFA-1 I-domain as the I-domain structure reveals,these data strongly suggest that the introduced Cys287 and Cys294 formeda disulfide bridge that constrains the I-domain in a high affinitystate.

Furthermore, the effect of divalent cations on ligand binding of theisolated I-domains expressed on the surface of K562 transfectants wastested. The binding reactions were performed in HEPES/NaCl/glucose (20mM HEPES, pH 7.5, 140 mM NaCl, 2 mg/ml glucose) supplemented with 1 mMMn²⁺, 1 mM Mg²⁺, or 1 mM EDTA. As shown in FIG. 8B, the binding of theK287C/K294C I-domain to ICAM-1 was divalent cation dependent, as EDTAtreatment abolished the binding. In contrast to intact wild-type LFA-1,Mn²⁺ or Mg²⁺ did not activate ligand binding of the isolated wild-typeI-domain or the mutant L289C/K294C I-domain.

The effect of the I-domain antibodies on ligand binding of the isolatedK287C/K294C I-domain was also examined. Transfectants expressing intactLFA-1 were pre-incubated with the activating antibody CBRLFA-1/2, andbinding of the cells to ICAM-1 was performed in the presence of theI-domain antibodies TS1/22, TS2/6, TS1/11, TS1/12, CBRLFA-1/9,CBRLFA-1/1, 25.3.1, TS2/14, or the nonbinding antibody X63, asindicated. Monoclonal antibodies TS1/22, TS2/6, TS1/11, TS1/12 andCBRLFA-1/9 inhibited binding of the isolated K287C/K294C I-domain toICAM-1, whereas antibodies 25-3-1, TS214 and CBRLFA-1/1 did not (FIG.8C). All antibodies, except for CBRLFA-1/1, bound to the mutantK287C/K294C I-domain as well as the wild-type I-domain as determined byflow cytometry. The binding of CBRLFA-1/1 to the mutant I-domain wasreduced to 80% of the wild-type I-domain. These results are consistentwith those obtained with the intact LFA-1 K287C/K294C mutant (Tables 2and 3), and indicate that the isolated K287C/K294C I-domain remainsstructural integrity as in the intact molecule.

Example 5 Inhibition of LFA-1 Function In Vitro and In Vivo by SolubleI-Domain Mutants

A soluble αL I-domain mutant stabilized in the open conformation by adisulfide bond (K287C/K294C) was made in E. coli.

Briefly, recombinant mutant αL I-domain stabilized in the openconformation (K287C/K294C), or recombinant wild-type αL I-domain fromamino acid residue G128 to Y307, were cloned into pET11b (Novagen) andexpressed in E. coli induced with 1 mM IPTG for 4 hours. The recombinantproteins were purified from inclusion bodies by solubilization ofinclusion bodies in 6M guanidine HCl and were refolded by dilution inthe presence of 0.1 mM Cu²⁺/phenanthrolin to enhance formation ofdisulfide bonds. Protein was concentrated by ammonium sulfateprecipitation, dialyzed, and purified over a monoQ ion-exchange column.To remove any material in which the disulfide bond did not form, freesulfhydryls were reacted with activated biotin and passed over astreptavidin column. The recombinant proteins were then purified by gelfiltration and concentrated by Centriprep. For BIAcore™ analysis,recombinant ICAM-1, ICAM-2 and ICAM-3 Fc chimeras were immobilized onthe BIAcore™ sensor chip by an amine-coupling method. Recombinant αLI-domains were flowed in, and BIAcore™ assays were performed withTris-buffered saline supplemented with 1 mM MgCl₂ or 2 mM EDTA, at aflow rate of 10 μl/minute at 25° C.

The purified open I-domain showed high affinity to its ligands, ICAM-1,-2, and -3, in the presence of 1 mM MgCl₂ as assessed by BIAcore™analysis, whereas binding of a soluble wild-type I domain was notdetectable (FIG. 9, Panels A, C and E; Table 4). The interaction of theopen I-domain with ligands was divalent cation-dependent, and wasabolished in the presence of 2 mM EDTA, suggesting that the interactiondepends on MIDAS. Since the wild-type I-domain showed no interactionwith ligands, the open I-domain allowed for the detailed analysis of thebinding kinetics of LFA-1 with its ligands. To analyze binding kinetics,different concentrations of open I-domain were tested for ligand binding(FIG. 9, Panels B, D and F). Kinetic analysis demonstrated a fastassociation rate (1.28×10⁵ M⁻¹ s⁻¹) and an intermediate dissociationrate (0.0230 s⁻¹) for ICAM-1, the major ligand on endothelial cells(Table 4). The K_(D) for ICAM-1 is in the nanomolar range and ICAM-1showed the highest affinity, followed by ICAM-2 and ICAM-3. The openI-domain also showed nanomolar range affinity for murine ICAM-1.

TABLE 4 Kinetics of open I-domain binding to ICAMs Ligand k_(on)(M⁻¹s⁻¹) k_(off) (s⁻¹) K_(D) (nM⁻¹) ICAM-1 1.28 × 10⁵ 0.0230 180 ICAM-20.23 × 10⁵ 0.0118 513 ICAM-3 0.19 × 10⁵ 0.0749 3942 k_(on), k_(off), andK_(D) were calculated based on 1:1 interaction model usingBIAevaluation ™ software.

In another study, measurements of the affinity of the recombinant;soluble high affinity αL I domain for its ligand ICAM-1 show a Kd of 200nM, as assessed by BIAcore. Thus, the isolated, high affinity conformerof the αL I domain is as active as the most activated αLβ2 heterodimer.

The activity of the soluble open I-domain to inhibit LFA-1-dependentadhesion was tested. In one study, K562 cells stably expressingwild-type LFA-1 were fluorescently labeled by BCECF and LFA-1 on thecell surface was activated by the activating monoclonal antibody,CBRLFA-1/2 in L15 media supplemented with FCS. The cells weresubsequently incubated in ICAM-1 coated 96-well plastic plates in thepresence or absence of I-domains. After incubation for 40 minutes at 37°C., unbound cells were washed off on a Microplate Autowasher. Thefluorescence content of total input cells and the bound cells in eachwell was quantitated on a Fluorescent Concentration Analyzer. The boundcells were expressed as a percentage of total input cells per samplewell. In contrast to the wild-type I-domain, the open I-domain mutantstrongly inhibited adhesion of LFA-1 expressing cells to immobilizedICAM-1 (FIG. 10A).

In another study, the murine T lymphoma cell line EL-4 which expressesboth murine LFA-1 and its ligands, including murine ICAM-1, and whichexhibits LFA-1-dependent homotypic aggregation upon activation by PMAwas used. Cells were incubated in a 96 well plate in the presence of 50ng/ml PMA and varying amounts of soluble I-domains. After incubation for2 hours at 37° C., 5% CO₂, the degree of aggregation was scored underthe microscope as follows: 0 indicated that essentially no cells wereclustered; 1 indicated that <10% of cells were aggregated; 2 indicatedclustering of <50%; 3 indicated that up to 100% of cells were in small,loose aggregates; 4 indicated that nearly 100% of cells were in largerclusters; and 5 indicated that nearly 100% of cells were in very large,tight clusters. As shown in FIG. 10B, the soluble open I-domain alsoinhibited PMA-induced LFA-1 dependent homotypic aggregation of themurine T-cell line EL-4.

Moreover, the ability of the open I-domain mutants to inhibit LFA-1function in vivo was tested by visualizing microcirculation in theperipheral lymph node (LN) with intravital microscopy. Briefly, a smallbolus (20–50 μl) of LN cell suspensions from T-GFP mice wereretrogradely injected through a femoral artery catheter and visualizedin the subiliac LN by fluorescent epi-illumination from avideo-triggered xenon arc stroboscope. After recording control T^(GFP)cell behavior in the absence of I-domain, the mouse was pretreated byintra-arterial injection of I-domain (10 μg/g of weight) 5 minutesbefore T^(GFP) cell injection. All scenes were recorded on videotape andoff-line analysis was done. The rolling fraction was calculated aspercentage of rolling cells amount the total number of T^(GFP) cellsthat entered a venule. The sticking (firm adhesion) fraction wasdetermined as the percentage of T^(GFP) cells becoming firmly adherentfor >20 seconds in the number of T^(GFP) cells that rolled in a venule.Results were semi-quantitatively scored as follows: −: 0%, ±: 0–5%, +:5–20%, ++: 20–40%, +++: 40–60%, ++++: 60–80%, +++++:80–100%.

As shown in Table 5, below, injection of the open I-domain but not thewild-type I-domain effectively blocked firm adhesion of T-lymphocytes tohigh endothelial venules, which is LFA-1-dependent. Lymphocyte rollingthat is mediated by L-selectin and PNAd was not compromised, suggestingthat the inhibitory effects of the open I-domain was LFA-1 specific.

TABLE 5 In vivo firm adhesion of lymphocytes under flow in peripherallymph node high endothelial venules was inhibited by open but notwild-type I-domain Fraction I-domain rolling firm adhesiontransmigration none +++ ++ ± wild-type +++ ++ ± open ++++ ± −Kinetics of the Binding of αL Mutant I-domains to ICAM-1

To further investigate the kinetics of the interaction of the αLI-domains with ICAM-1, recombinant soluble αL I-domains were expressedin E. coli, refolded and purified. As shown in Table 6, below, theaffinity of E284C/E301C is nearly comparable to K287C/K294C. Theaffinity of L161C/F299C, K160C/F299C, and L161C/T300C are significantlyhigher than wild type, but 20–30 times lower than high-affinity open αLI-domain, K287C/K294C. L161C/F299C, K160C/F299C, and L161 C/T300C arereferred to as intermediate-affinity αL I-domains.

TABLE 6 Kinetics of interaction of αL I-domains with ICAM-1 Kon αLI-domain (1/Ms) Koff (1/s) KD (μM) Locked open K287C/K294C 1.28 × 10⁵0.0230 0.180 E284C/E301C 1.28 × 10⁵ 0.0459 0.360 L161C/F299C 1.36 × 10⁵0.513 3.76 K160C/F299C 1.53 × 10⁵ 0.67 4.39 L161C/T300C 1.35 × 10⁵ 0.654.8 WT 2.22 × 10³ 3.00 1350 Locked closed L289C/K294C 2.11 × 10³ 2.841760 Recombinant soluble αL I-domains were expressed in E. coli,refolded and purified. Kinetics of binding of the I-domains to ICAM-1was measured by BIAcore ™ instruments. Kinetics was analyzedBIAevaluation ™ software. KD was calculated by Scatchard plots usingdata at steady states. Koff was obtained by curve fitting of thedissociation phase using 1:1 binding model. Kon was calculated byKoff/KD.

Example 6 Construction and Activity of Mac-1 Cysteine SubstitutionMutants

A similar approach was taken to design an open, high affinityconformation of Mac-1 by introducing a disulfide bond into the I-domain.The design of Mac-1 cysteine substitution mutants was described inExample 1.

TABLE 7 Cα and Cβ between mutated residues in either open or closedconformation ido (open jlm (closed conformation) conformation) mutationsCα Cβ Cα Cβ Locked open Q163C/Q309C 8.37 6.36 9.11 7.16 Q298C/N301C 5.314.21 9.05 10.91 D294C/T307C 9.21 8.67 16.01 17.52 D294C/Q311C 9.02 7.089.79 10.02 F297C/A304C 6.31 3.78 11.18 10.17 Locked closed Q163C/R313C13.8 13.33 7.36 5.15 The distance between wild-type residues wasmeasured by Look ™ software in open conformation (1 ido) or closedconformation (1 jlm).

In order to assess the effect of the introduction of pairs ofpotentially disulfide bond-forming cysteines into the I-domain of αMβ2on CBRM1/5 activation-dependent epitope expression and ligand binding,plasmids encoding the wild-type or mutant αM subunits and the β2 subunitwere co-transfected into 293T and K562 cells. αβ heterodimer formationwas confirmed using monoclonal antibody CBRM1/32 which recognizes anepitope in the putative β-propeller domain of the αM subunit only afterassociation with the β2 subunit, and antibody CBRM1/5 was used to detectintegrin activation.

The Q163C/Q309C pair of mutations worked well (FIG. 11B, FIGS. 12B andC). This mutant introduces a putative disulfide bond near the bottomfront of the I-domain, between residues that are in the lower one-thirdof the last α-helix and the first α-helix, and have Cβ carbons that are6.36 Å apart in the 1ido structure. In contrast, the Cβ carbons for theD294C/T307C and D294C/N311C substitutions are 8.67 Å and 7.08 Å apart,respectively. The Cβ carbons for the Q298C/N301C and F297C/A304Csubstitutions are within the ideal range, however these substitutionsare closer to the loop between the last β-strand and α-helix, and musthave unfavorable effects such as distorting the ligand binding site.

When expressed within an intact heterodimer in transiently transfected293T cells, the Q163C/Q309C mutant is expressed half as well aswild-type as measured by CBRM1/32 antibody, but the ratio of the CBRM1/5activation-dependent epitope to CBRM1/32 expression is markedly higher(FIG. 11A). In addition, the adhesion of 293T cells expressing the Mac-1Q163C/Q309C mutant to iC3b coated on plastic, as assayed in L15/FBSmedium at room temperature, was higher than wild-type, despite its lowerexpression (FIG. 11B).

Alternatively, isolated Mac-1 mutant I-domains were expressed on thecell surface in conjunction with an artificial signal sequence andtransmembrane domain of the PDGF receptor. Adhesion was assayed inL15/FBS/MnCl₂ at 37° C. The isolated wild-type I-domain showed nobinding to iC3b, whereas the previously described mutants withcomputationally redesigned hydrophobic cores, ido 1r and ido2r, wereactive (FIG. 11C) (Shimaoka, M et al. (2000) Nature Structural Biology7:674–678). The Q163C/Q309C mutant I-domain exhibited strong specificligand binding that was completely blocked by the inhibitory I-domainmonoclonal antibody CBRM1/5 (FIG. 12C).

In a further study, the open I-domain mutants Q163C/Q309C andD294C/Q311C were stably expressed in K562 cells, and clones expressingthe same levels of receptors were selected. Adhesion assays toimmobilized iC3b were performed with L15/FBS at 37° C. In contrast to293T cells, wild-type Mac-1 has little basal activity for ligand bindingin these cells (FIGS. 12A and 12B). Both Q163C/Q309C and D294C/Q311Cshowed increased CBRM1/5 activation-dependent epitope expression andincreased ligand binding when expressed in an intact αMβ2 heterodimer,as compared to wild-type (FIGS. 12A and 12B). Moreover, K562 cellsexpressing isolated open I-domain mutants on the cell surface showedstrong specific binding to iC3b as compared to wild-type (FIG. 12C).

In order to confirm that the increased ligand binding activity of theopen I-domain mutants is induced by the formation of a disulfide bond,the effect of the reducing agent DTT was tested. Binding of αMβ2transfectants containing mutant I-domains to immobilized iC3b on plasticwas tested in the presence and absence of DTT. As summaried in Table 8,below, locked open αM I-domains, (Q163C/Q309C) and (D294C/Q311c), areactive in the absence of activation and their activities are partlyreduced by disulfide reduction by DTT. By contrast, locked closed αMI-domain Q163C/R313C is inactive and resistant to activation, butbecomes activatable after disulfide reduction by DTT.

As shown in FIG. 12C, DTT treatment abolished ligand binding by isolatedlocked open I-domains. In contrast, DTT increased binding of the intactwild-type αMβ2 (FIG. 2B), indicating that DTT used in this experimentwas not toxic and abolishment of ligand binding by the open I-domainmutants was not due to a non-specific effect of DTT. Taken together,these data suggest that the introduced cysteines result in the formationof a disulfide bridge that constrains the Mac-1 I-domain in an open orclosed conformation.

TABLE 8 Summary of adhesion assay of αMβ2 transfectant containing mutantI-domains −DTT −DTT +DTT +DTT mutations −activation +activation−activation +activation Wild type ± ++++ ++ ++++ Locked open Q163C/Q309C++++ ++++ ++ ++++ Q298C/N301C ± + NT NT D294C/T307C ± + NT NTD294C/Q311C ++++ ++++ ++ ++++ F297C/A304C ± ++ NT NT Locked closedQ163C/R313C ± ± ++ +++ Binding of αMβ2 transfectants containing mutantI-domains to immobilized iC3b on plastic was tested. Results weresemi-quantitatively scored as follow; ±: 0–5%, +: 5–25%, ++ 25–50%, +++:50–75%, ++++: 75–100% of binding by activated wild type transfectant.NT: not tested DTT: disulfide reduction by DTT treatment. +activation:activated by activating mAB CBR LFA-1/2

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. The modified integrin I-domain polypeptide of SEQ ID NO:2 containingamino acid substitutions selected from the group consisting ofK287C/K294C, E284C/E301C, L161C/F299C, K160C/F299C, L161C/T300C andL289C/K294C, said substitutions being based on the mature form of thepolypeptide.
 2. A modified integrin I-domain polypeptide of claim 1which is stabilized in the open conformation.
 3. A modified integrinI-domain polypeptide of claim 1 which is stabilized in the closedconformation.
 4. A modified integrin I-domain polypeptide of claim 2which binds ligand with high affinity.
 5. A modified integrin I-domainpolypeptide of claim 1 which is comprised within an integrin αL subunit.6. A modified integrin I-domain polypeptide of claim 5 which is furtherassociated with an integrin β2 subunit.
 7. A modified integrin I-domainpolypeptide of claim 1 which is a soluble polypeptide.
 8. A modifiedintegrin I-domain polypeptide of claim 1 which is operatively linked toa heterologous polypeptide.
 9. A composition comprising a modifiedintegrin I-domain polypeptide of claim 1 and a pharmaceuticallyacceptable carrier.
 10. A composition of claim 9, further comprising ananti-inflammatory or immunosuppressive agent.